Cysteine protease

ABSTRACT

The present invention relates to a novel polypeptide which displays IgG cysteine protease activity, and in vivo and ex vivo uses thereof. Uses of the polypeptide include methods for the prevention or treatment of diseases and conditions mediated by IgG, and methods for the analysis of IgG.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. Non-Provisional applicationSer. No. 15/550,309 filed Aug. 10, 2017, which is a national phaseapplication under 35 U.S.C. § 371 that claims priority to InternationalApplication No. PCT/EP2016/053052 filed Feb. 12, 2016, which claimspriority to Great Britain Patent Application No. 1502306.2, Feb. 12,2015, all of which are incorporated herein by reference in theirentirety.

INCORPORATION OF SEQUENCE LISTING

The instant application contains a Sequence Listing, named“SL_KEMP_P0066USC1_1001125962_N404707USA.txt” (68,370 bytes) which hasbeen submitted electronically in ASCII format and is hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel polypeptide which displays IgGcysteine protease activity, and in vivo and ex vivo uses thereof. Usesof the polypeptide include methods for the prevention or treatment ofdiseases and conditions mediated by IgG, and methods for the analysis ofIgG.

BACKGROUND OF THE INVENTION

IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes) is anextracellular cysteine protease produced by the human pathogen S.pyogenes. IdeS was originally isolated from a group A Streptococcusstrain of serotype Ml, but the ides gene has now been identified in alltested group A Streptococcus strains. IdeS has an extraordinarily highdegree of substrate specificity, with its only identified substratebeing IgG. IdeS catalyses a single proteolytic cleavage in the lowerhinge region of the heavy chains of all subclasses of human IgG. IdeSalso catalyses an equivalent cleavage of the heavy chains of somesubclasses of IgG in various animals. IdeS efficiently cleaves IgG to Fcand F(ab′)₂ fragments via a two-stage mechanism. In the first stage, one(first) heavy chain of IgG is cleaved to generate a single cleaved IgG(scIgG) molecule with a non-covalently bound Fc molecule. The scIgGmolecule is effectively an intermediate product which retains theremaining (second) heavy chain of the original IgG molecule. In thesecond stage of the mechanism this second heavy chain is cleaved by IdeSto release a F(ab′)2 fragment and a homodimeric Fc fragment. These arethe products generally observed under physiological conditions. Underreducing conditions the F(ab′)2 fragment may dissociate to two Fabfragments and the homodimeric Fc may dissociate into its componentmonomers.

SUMMARY OF THE INVENTION

The IgG cleaving ability of IdeS has been shown to have utility ex vivo,for example in methods for production of Fab and Fc fragments, which maybe used for the analysis of IgG. See, for example, WO2003051914 andWO2009033670. IdeS has also been shown to have in vivo utility as atherapeutic agent, since it is capable of the in vivo cleavage of IgGmolecules which mediate disease or which are otherwise undesirable. See,for example, WO2003051914, WO2006131347 and WO2013110946. IdeS may beused as a therapy for any disease or condition wholly or partly mediatedby IgG. Many autoimmune diseases are wholly or partly mediated by IgG,as is the acute rejection of donated organs.

However, IdeS is an immunogenic protein. That is, when IdeS is used as atherapeutic agent the immune system of the subject receiving IdeS willoften respond to it. The reaction of the immune system to IdeS willtypically involve the production of antibodies specific for IdeS. Theseantibodies may be referred to herein as anti-drug antibodies (ADA)specific for IdeS or “IdeS-specific ADA”. The immune response to IdeS ingeneral, and the production of IdeS-specific ADA in particular, maycause two related types of problem. Firstly, the efficacy of IdeS may bereduced, e.g. due to ADA binding, potentially requiring higher or repeatdoses to achieve the same effect. ADA which have this effect may bereferred to as “neutralising ADA”. Secondly, there may be undesirable oreven harmful complications, such as a hyper-inflammatory responsetriggered by immune complexes of ADA and IdeS. The higher the quantityof ADA specific for IdeS in a given subject, the greater the likelihoodof these problems. The presence and quantity of IdeS-specific ADAmolecules in a patient may be determined by any suitable method, such asan agent specific CAP FEIA (ImmunoCAP) test or a titre assay conductedon a serum sample from the patient. Above a threshold determined by theclinician, the quantity of IdeS-specific ADA molecules in the patientmay preclude administration of IdeS, or indicate that a higher dose ofIdeS is required. Such a higher dose may in turn result in an increasedquantity of IdeS-specific ADA molecules in the patient, therebyprecluding further administration of IdeS.

IdeS is a virulence factor of S. pyogenes, which is responsible forcommon infections like tonsillitis and strep throat. Accordingly mosthuman subjects have encountered IdeS in this context and are likely tohave anti-IdeS antibodies in the bloodstream. IdeS-specific ADA areroutinely detected in serum samples from random human subjects (likelydue to prior streptococcal infections), as well as in IVIg (IntravenousImmunoglobulin) preparations, which are preparations of IgG extractedfrom the pooled serum of thousands of donors. Even if a subject does notpossess IdeS-specific ADA prior to an initial administration of IdeS, itis likely that such molecules will be produced subsequently. Thus, forany given subject, the problems associated with the immunogenicity ofIdeS are likely to present a barrier to the use of IdeS as a treatment.These problems may require increases to the dose of IdeS and/or precludetreatment with IdeS entirely, particularly if repeat administrations arerequired. Existing approaches to problems of this type involve, forexample, PEGylation of a therapeutic agent to reduce immunogenicity orco-administration of the therapeutic agent with an immune-suppressiveagent.

The present inventors have adopted an entirely different approach. Theinventors have identified specific positions within the sequence of IdeSwhich, when modified as described herein, lead to novel polypeptides forwhich the problems associated with immunogenicity are reduced ascompared to IdeS. Some modifications may increase the efficacy atcleaving IgG of the polypeptide of the invention relative to IdeS,thereby indirectly reducing immunogenicity by permitting the use of alower dose or concentration to achieve the same effect. Alternatively,or in addition, other modifications may directly reduce immunogenicityby reducing the ability of IdeS-specific antibodies to recognise thepolypeptide of the invention relative to IdeS.

The full sequence of IdeS is publically available as NCBI ReferenceSequence no WP_010922160.1 and is provided herein as SEQ ID NO: 1. Thissequence includes an N terminal methionine followed by a 28 amino acidsecretion signal sequence. The N terminal methionine and the signalsequence (a total of 29 amino acids at the N terminus) are typicallyremoved to form the mature IdeS protein, the sequence of which ispublically available as Genbank accession no. ADF13949.1 and is providedherein as SEQ ID NO: 2.

Unless otherwise stated, all references to numbering of amino acidpositions in the polypeptides disclosed herein is based on the numberingof the corresponding positions in SEQ ID NO: 1, starting from the Nterminus. Thus, since SEQ ID NO: 2 lacks the N terminal methionine and28 amino acid signal sequence of SEQ ID NO: 1, the aspartic acid (D)residue at the N terminus of SEQ ID NO: 2 is referred to as position 30as this the corresponding position in SEQ ID NO: 1. Applying thisnumbering scheme, the most critical residue for IgG cysteine proteaseactivity of IdeS is the cysteine (C) at position 94 (65^(th) residuefrom the N terminus of SEQ ID NO: 2). Other residues likely to beimportant for IgG cysteine protease activity are the lysine (K) atposition 84, the histidine (H) at position 262, and the aspartic acid(D) at each of positions 284 and 286. These are the 55^(th), 233^(rd),255^(th) and 257^(th) residues from the N terminus of SEQ ID NO: 2,respectively.

In accordance with the present invention, there is thus provided apolypeptide having IgG cysteine protease activity and comprising avariant of the sequence of SEQ ID NO:2, which variant:

-   -   (a) is at least 50% identical to SEQ ID NO: 2;    -   (b) has a cysteine (C) at the position in said variant sequence        which corresponds to position 94 of SEQ ID NO: 1; and optionally    -   (c) has, at the positions in said variant sequence which        correspond to positions 84, 262, 284 and 286 of SEQ ID NO: 1, a        lysine (K), a histidine (H), an aspartic acid (D) and an        aspartic acid (D), respectively;

wherein said polypeptide is more effective at cleaving IgG than IdeSand/or is less immunogenic than IdeS. The polypeptide of the inventionmay be more effective at cleaving IgG1 than IgG2.

Preferably said variant of SEQ ID NO: 2:

-   -   (1) has a positively charged amino acid at the position in said        variant which corresponds to position 130 of SEQ ID NO: 1,        optionally wherein said positively charged amino acid is        arginine (R) or lysine (K); and/or    -   (2) has a positively charged amino acid at the position in said        variant which corresponds to position 131 of SEQ ID NO: 1,        optionally wherein said positively charged amino acid is        arginine (R) or lysine (K); and/or    -   (3) does not include the contiguous sequence NQTN; and/or    -   (4) does not include the contiguous sequence DSFSANQEIR        YSEVTPYHVT.

The invention also provides a polynucleotide, an expression vector or ahost cell encoding or expressing a polypeptide of the invention.

The invention also provides a method of treating or preventing a diseaseor condition mediated by IgG antibodies in a subject, the methodcomprising administering to the subject a therapeutically orprophylactically effective amount of a polypeptide of the invention. Themethod may typically comprise multiple administrations of saidpolypeptide to the subject.

The invention also provides a method of treating, ex vivo, blood takenfrom a patient, typically a patient suffering from a disease orcondition mediated by IgG antibodies, which method comprises contactingthe blood with a polypeptide of the invention.

The invention also provides a method for improving the benefit to asubject of a therapy or therapeutic agent, the method comprising (a)administering to the subject a polypeptide of the invention; and (b)subsequently administering said therapy or said therapeutic agent to thesubject; wherein:

-   -   said therapy is an organ transplant or said therapeutic agent is        an antibody, a gene therapy such as a viral vector, a        replacement for a defective endogenous factor such as an enzyme,        a growth or a clotting factor, or a cell therapy;    -   the amount of said polypeptide administered is sufficient to        cleave substantially all IgG molecules present in the plasma of        the subject; and    -   steps (a) and (b) are separated by a time interval which is        sufficient to cleave substantially all IgG molecules present in        the plasma of the subject.

The invention also provides a method of generating Fc, Fab or F(ab′)₂fragments of IgG comprising contacting IgG with a polypeptide of theinvention, preferably ex vivo.

Also provided are kits for carrying out the methods according to theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of a representative assay to determine thepotency (efficacy at cleavage of IgG) of polypeptides of the inventionas compared to controls.

FIG. 2 shows the results of a representative SDS-PAGE gel used tovisualize the cleavage products produced by incubation of IgG1 withpolypeptides of the invention or controls.

FIG. 3 shows the results of a representative SDS-PAGE gel used tovisualize the cleavage products produced by incubation of IgG1 withfurther polypeptides of the invention or controls.

FIG. 4 shows the results of a representative SDS-PAGE gel used tovisualize the cleavage products produced by incubation of IgG2 withpolypeptides of the invention or controls.

FIGS. 5 and 6 show the results of representative competition assays todetermine the level of recognition of polypeptides of the invention byIdeS-specific antibodies, as compared to controls.

FIG. 7 shows representative titration curves for cleavage of IgG1 bydifferent IgG cysteine protease polypeptides.

FIG. 8 shows representative titration curves for cleavage of IgG2 bydifferent IgG cysteine protease polypeptides.

FIG. 9 shows the results of a representative SDS-PAGE used to visualizethe cleavage products produced by incubation of IgG with polypeptides ofthe invention or controls.

FIG. 10 shows the results of a representative SDS-PAGE used to visualizethe cleavage products produced by incubation of IgG with polypeptides ofthe invention or controls.

FIG. 11 shows the results of a representative SDS-PAGE used to visualizethe cleavage products produced by incubation of IVIg with polypeptidesof the invention or controls.

FIG. 12 shows the results of a representative SDS-PAGE used to visualizethe cleavage products produced by incubation of IVIg with polypeptidesof the invention or controls.

FIG. 13 Schematic representation of the cleavage of immunoglobulins bypolypeptides of the invention.

FIG. 14 shows the results of a representative % competition of ADAbinding sites with polypeptides of the invention or controls.

FIG. 15 shows the results of a further representative % competition ofADA binding sites with polypeptides of the invention or controls.

FIG. 16 shows the results of a representative efficacy ELISA used todetermine the efficacy of the polypeptides of the invention in cleavinghuman IgG in vivo.

FIG. 17 shows the results of a representative SDS-PAGE use to visualizethe IgG cleavage products produced in vivo by polypeptides of theinvention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the full sequence of IdeS including N terminalmethionine and signal sequence. Also disclosed as NCBI ReferenceSequence no WP_010922160.1

SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N terminalmethionine and signal sequence. Also disclosed as Genbank accession no.ADF13949.1

SEQ ID NOs: 3 to 16 are the sequences of exemplary polypeptides of theinvention

SEQ ID NO: 17 is the sequence of an IdeS polypeptide used herein as acontrol. Comprises the sequence of SEQ ID NO: 2 with an additional Nterminal methionine and a histidine tag (internal reference pCART124).

SEQ ID NO: 18 is the contiguous sequence NQTN, which corresponds topositions 336-339 of SEQ ID NO: 1.

SEQ ID NO: 19 is the contiguous sequence DSFSANQEIR YSEVTPYHVT, whichcorresponds to positions 30-49 of SEQ ID NO: 1.

SEQ ID NOs: 20 to 34 are nucleotide sequences encoding polypeptidesdisclosed herein.

SEQ ID NO: 35 is the sequence SFSANQEIRY SEVTPYHVT, which corresponds topositions 31-49 of SEQ ID NO: 1.

SEQ ID NO: 36 is the sequence DYQRNATEAY AKEVPHQIT, which corresponds topositions 36-54 of the IdeZ polypeptide NCBI Reference Sequence noWP_014622780.1.

SEQ ID NO: 37 is the sequence DDYQRNATEA YAKEVPHQIT, which may bepresent at the N terminus of a polypeptide of the invention.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolypeptide” includes “polypeptides”, and the like.

A “polypeptide” is used herein in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The term “polypeptide” thus includes shortpeptide sequences and also longer polypeptides and proteins. As usedherein, the term “amino acid” refers to either natural and/or unnaturalor synthetic amino acids, including both D or L optical isomers, andamino acid analogs and peptidomimetics.

The terms “patient” and “subject” are used interchangeably and typicallyrefer to a human. References to IgG typically refer to human IgG unlessotherwise stated.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Functional Features of the Polypeptide

The present invention relates to a novel polypeptide having IgG cysteineprotease activity, wherein said polypeptide is more effective atcleaving IgG than IdeS and/or is less immunogenic than IdeS. In thecontext of a control or a comparison relative to a polypeptide of theinvention, “IdeS” refers to a polypeptide consisting of the amino acidsequence of SEQ ID NO: 2. Alternatively or in addition, “IdeS” when usedas a control or a comparison may refer to a polypeptide comprising thesequence the amino acid sequence of SEQ ID NO: 2 with an additionalmethionine (M) residue at the N terminus and/or a tag at the C terminusto assist with expression in and isolation from standard bacterialexpression systems. Suitable tags include a histidine tag which may bejoined directly to the C terminus of a polypeptide or joined indirectlyby any suitable linker sequence, such as 3, 4 or 5 glycine residues. Thehistidine tag typically consists of six histidine residues, although itcan be longer than this, typically up to 7, 8, 9, 10 or 20 amino acidsor shorter, for example 5, 4, 3, 2 or 1 amino acids. The sequence of anexemplary IdeS polypeptide used herein is a control is provided as SEQID NO: 14. This polypeptide comprises the sequence of SEQ ID NO: 2 withan additional N terminal methionine and a histidine tag and may bereferred to herein as pCART124.

IgG cysteine protease activity may be assessed by any suitable method,for example by incubating a polypeptide with a sample containing IgG anddetermining the presence of IgG cleavage products. Efficacy may beassessed in the presence or absence of an inhibitor, such as aneutralising antibody. However, efficacy herein will typically meanefficacy as assessed in the absence of such an inhibitor unlessotherwise stated. Suitable methods are described in the Examples. Theefficacy of a polypeptide at cleavage of IgG may be referred to hereinas the “potency” of the polypeptide. The potency of a polypeptide of theinvention is typically at least 1.5 fold, 2.0 fold, 2.5 fold, 3.0 fold,4.0 fold, 4.5 fold, 5.0 fold, 6.0 fold, 7.0 fold or 7.5 fold greaterthan the potency of IdeS measured in the same assay. The potency of apolypeptide of the invention is preferably at least 4.5 fold, morepreferably at least 6.0 fold and most preferably at least 7.5 foldgreater than the potency of IdeS measured in the same assay. Increasedpotency relative to IdeS is a desirable improvement irrespective of theproblems associated with immunogenicity of IdeS. However, such increasedpotency will typically also enable the use of a lower dose of apolypeptide of the invention for the same therapeutic effect as a higherdose of IdeS. The lower dose may also permit a greater number of repeatadministrations of a polypeptide of the invention relative to IdeS. Thisis because the use of a lower dose reduces the problems associated withimmunogenicity of a therapeutic agent, because the immune system is lesslikely to respond, or will respond less vigorously, to an agent which ispresent at a lower concentration. A polypeptide of the invention maytherefore be as immunogenic as IdeS or even more immunogenic than IdeSwhen present at an equivalent dose, but problems associated with thisimmunogenicity are reduced or avoided because a lower dose is requiredto achieve the same therapeutic effect. In an alternative embodiment, apolypeptide of the invention may have equivalent potency to IdeSprovided it is less immunogenic than IdeS when present at an equivalentdose.

Assays for assessing the efficacy of a polypeptide at the cleavage ofIgG, that is assays for assessing the potency of a polypeptide, are wellknown in the art and any suitable assay may be used. Suitable assaysinclude an ELISA-based assay, such as that which is described in theExamples. In such an assay, the wells of an assay plate will typicallybe coated with an antibody target, such as bovine serum albumin (BSA).Samples of the polypeptide to be tested are then added to the wells,followed by samples of target-specific antibody that is specific for BSAin this example. The polypeptide and antibody are allowed to interactunder conditions suitable for IgG cysteine protease activity. After asuitable interval, the assay plate will be washed and a detectorantibody which specifically binds to the target-specific antibody willbe added under conditions suitable for binding to the target-specificantibody. The detector antibody will bind to any intact target-specificantibody that has bound to the target in each well. After washing, theamount of detector antibody present in a well will be proportional tothe amount of target-specific antibody bound to that well. The detectorantibody may be conjugated directly or indirectly to a label or anotherreporter system (such as an enzyme), such that the amount of detectorantibody remaining in each well can be determined. The higher thepotency of the tested polypeptide that was in a well, the less intacttarget-specific antibody will remain and thus there will be lessdetector antibody. Typically, at least one well on a given assay platewill include IdeS instead of a polypeptide to be tested, so that thepotency of the tested polypeptides may be directly compared to thepotency of IdeS. The polypeptide of the invention may be more effectiveat cleaving IgG1 than IgG2.

Other assays may determine the potency of a tested polypeptide bydirectly visualizing and/or quantifying the fragments of IgG whichresult from cleavage of IgG by a tested polypeptide. An assay of thistype is also described in the Examples. Such an assay will typicallyincubate a sample of IgG with a test polypeptide (or with IdeS as acontrol) at differing concentrations in a titration series. The productswhich result from incubation at each concentration are then separatedusing gel electrophoresis, for example by SDS-PAGE. Whole IgG and thefragments which result from cleavage of IgG can then be identified bysize and quantified by the intensity of staining with a suitable dye.The greater the quantity of cleavage fragments, the greater the potencyof a tested polypeptide at a given concentration. A polypeptide of theinvention will typically produce detectable quantities of cleavagefragments at a lower concentration (a lower point in the titrationseries) than IdeS. This type of assay may also enable the identificationof test polypeptides that are more effective at cleaving the first orthe second heavy chain of an IgG molecule, as the quantities of thedifferent fragments resulting from each cleavage event may also bedetermined. This type of assay may also be adapted to determine theextent to which the presence of IdeS-specific ADA may reduce the potencyof a polypeptide of the invention. In the adapted assay, when a sampleof IgG is incubated with a test polypeptide (or with IdeS as a control),serum or an IVIg preparation containing IdeS-specific ADA is includedwith the reaction medium. Preferably, the potency of a polypeptide ofthe invention is not affected by the presence of ADA or is less reducedby the presence of ADA than the potency of IdeS in the same assay. Inother words, preferably the neutralizing effect of IdeS-specific ADA onthe polypeptide of the invention is the same or lower than theneutralizing effect of IdeS-specific ADA on IdeS, measured in the sameassay.

As indicated above, a polypeptide of the invention may be as immunogenicas IdeS or even more immunogenic than IdeS when present at an equivalentdose, because the problems associated with this immunogenicity arereduced or avoided since a lower dose of the polypeptide of theinvention is required to achieve the same therapeutic effect. However,typically a polypeptide of the invention is no more immunogenic thanIdeS and preferably it is less immunogenic than IdeS. That is, apolypeptide of the invention may result in the same or preferably alower immune response than IdeS when present at an equivalent dose orconcentration and measured in the same assay. The immunogenicity of apolypeptide of the invention is typically no more than 90%, no more than85%, no more than 80%, no more than 70%, no more than 60%, or no morethan 50% of the immunogenicity of IdeS measured in the same assay.Preferably the immunogenicity of a polypeptide of the invention is nomore than 85% of the immunogenicity of IdeS measured in the same assay.More preferably the immunogenicity of a polypeptide of the invention isno more than 70% of the immunogenicity of IdeS measured in the sameassay.

Assays for assessing the immunogenicity of a polypeptide are known inthe art and any suitable assay may be used. Preferred assays forassessing the immunogenicity of a polypeptide relative to theimmunogenicity of IdeS involves assessing the extent to which ADAspecific for IdeS also bind to a polypeptide of the invention. Assays ofthis type are described in the Examples.

One such an assay involves testing for competition between IdeS and atest polypeptide for binding to IdeS-specific ADA. Typically, the wellsof an assay plate are coated with IdeS, followed by administration of apre-incubated mixture of a solution containing IdeS-specific ADA, e.g.an IVIg preparation, and a test polypeptide (or IdeS as a control). Thepre-incubation takes place in the presence of an inhibitor of IgGcysteine protease activity, e.g. iodoacetic acid (IHAc), and at highsalt concentration so that only high affinity binding between proteinand ADA is permitted. The pre-incubated mixture is allowed to interactwith the IdeS coated wells. Any IdeS-specific ADA not bound to testpolypeptide will bind to the IdeS on the wells. After a suitableinterval, the assay plate will be washed and a detector antibody whichspecifically binds to IgG will be added under conditions suitable forbinding. The detector antibody will bind to any ADA that has bound tothe IdeS in each well. After washing, the amount of detector antibodypresent in a well will be inversely proportional to the amount of ADAthat had bound to the test polypeptide. The detector antibody may beconjugated directly or indirectly to a label or another reporter system(such as an enzyme), such that the amount of detector antibody remainingin each well can be determined. Typically, at least one well on a givenassay plate will be tested with a pre-incubated mixture of IVIg and IdeSinstead of a polypeptide to be tested, so that the binding of ADA to thetested polypeptides may be directly compared to the binding to IdeS.

Another suitable assay involves testing the extent to which a titrationseries of different concentrations of IdeS-specific ADA, e.g. an IVIgpreparation, binds to a test polypeptide as compared to IdeS.Preferably, a polypeptide of the invention will require a higherconcentration of ADA for binding to be detectable, relative to theconcentration of ADA for which binding to IdeS is detectable. Such anassay is described in the Examples. Such an assay typically involvescoating the wells of an assay plate with test polypeptide or IdeS,followed by incubating with each well with a different concentration ofIdeS-specific ADAfrom a titration series. The incubations are conductedin the presence of an inhibitor of IgG cysteine protease activity, e.g.iodoacetic acid (IHAc), and at high salt concentration so that only highaffinity binding between protein and ADA is permitted. After a suitableinterval, the assay plate will be washed and a detector antibody whichspecifically binds to IgG F(ab′)₂ will be added under conditionssuitable for binding. The detector antibody will bind to any ADA thathas bound to the test polypeptide or the IdeS in each well. Afterwashing, the amount of detector antibody present in a well will bedirectly proportional to the amount of ADA that had bound to the testpolypeptide or IdeS. The detector antibody may be conjugated directly orindirectly to a label or another reporter system (such as an enzyme),such that the amount of detector antibody remaining in each well can bedetermined. At least one well on a given assay plate will be incubatedwith buffer lacking ADA as a blank to establish a threshold level fordetection of binding in the test wells.

Structural Features of the Polypeptide

This section sets out the structural features of a polypeptide of theinvention, which apply in addition to the functional features outlinedin the preceding section.

The polypeptide of the invention is typically at least 100, 150, 200,250, 260, 270, 280, 290 or 300 amino acids in length. The polypeptide ofthe invention is typically no larger than 400, 350, 340, 330, 320 or 310amino acids in length. It will be appreciated that any of the abovelisted lower limits may be combined with any of the above listed upperlimits to provide a range for the length the polypeptide of theinvention. For example, the polypeptide may be 100 to 400 amino acids inlength, or 250 to 350 amino acids in length. The polypeptide ispreferably 290 to 320 amino acids in length, most preferably 300 to 310amino acids in length.

The primary structure (amino acid sequence) of a polypeptide of theinvention is based on the primary structure of IdeS, specifically theamino acid sequence of SEQ ID NO: 2. The sequence of a polypeptide ofthe invention comprises a variant of the amino acid sequence of SEQ IDNO: 2 which is at least 50% identical to the amino acid sequence of SEQID NO: 2. The variant sequence may be at least 60%, at least 70%, atleast 80%, at least, 85%, preferably at least 90%, at least 95%, atleast 98% or at least 99% identical to the sequence of SEQ ID NO:2. Thevariant may be identical to the sequence of SEQ ID NO: 2 apart from theinclusion of one or more of the specific modifications identifiedherein. Identity relative to the sequence of SEQ ID NO: 2 can bemeasured over a region of at least 50, at least 100, at least 200, atleast 300 or more contiguous amino acids of the sequence shown in SEQ IDNO: 2, or more preferably over the full length of SEQ ID NO: 2.

Amino acid identity may be calculated using any suitable algorithm. Forexample the PILEUP and BLAST algorithms can be used to calculateidentity or line up sequences (such as identifying equivalent orcorresponding sequences (typically on their default settings), forexample as described in Altschul S. F. (1993) J Mol Evol 36:290-300;Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pair(HSPs) by identifying short words of length W in the query sequence thateither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighbourhood word score threshold (Altschul et al,supra). These initial neighbourhood word hits act as seeds forinitiating searches to find HSPs containing them. The word hits areextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extensions for the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a word length (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between twopolynucleotide or amino acid sequences would occur by chance. Forexample, a sequence is considered similar to another sequence if thesmallest sum probability in comparison of the first sequence to thesecond sequence is less than about 1, preferably less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001. Alternatively, the UWGCG Package provides the BESTFITprogram which can be used to calculate identity (for example used on itsdefault settings) (Devereux et al (1984) Nucleic Acids Research 12,387-395).

The sequence of a polypeptide of the invention comprises a variant ofthe amino acid sequence of SEQ ID NO: 2 in which modifications, such asamino acid additions, deletions or substitutions are made relative tothe sequence of SEQ ID NO: 2. Unless otherwise specified, themodifications are preferably conservative amino acid substitutions.Conservative substitutions replace amino acids with other amino acids ofsimilar chemical structure, similar chemical properties or similarside-chain volume. The amino acids introduced may have similar polarity,hydrophilicity, hydrophobicity, basicity, acidity, neutrality or chargeto the amino acids they replace. Alternatively, the conservativesubstitution may introduce another amino acid that is aromatic oraliphatic in the place of a pre-existing aromatic or aliphatic aminoacid. Conservative amino acid changes are well-known in the art and maybe selected in accordance with the properties of the 20 main amino acidsas defined in Table A1 below. Where amino acids have similar polarity,this can be determined by reference to the hydropathy scale for aminoacid side chains in Table A2.

TABLE A1 Chemical properties of amino acids Ala (A) aliphatic,hydrophobic, neutral Met (M) hydrophobic, neutral Cys (C) polar,hydrophobic, neutral Asn (N) polar, hydrophilic, neutral Asp (D) polar,hydrophilic, charged (−) Pro (P) hydrophobic, neutral Glu (E) polar,hydrophilic, charged (−) Gln (Q) polar, hydrophilic, neutral Phe (F)aromatic, hydrophobic, neutral Arg (R) polar, hydrophilic, charged (+)Gly (G) aliphatic, neutral Ser (S) polar, hydrophilic, neutral His (H)aromatic, polar, hydrophilic, charged (+) Thr (T) polar, hydrophilic,neutral Ile (I) aliphatic, hydrophobic, neutral Val (V) aliphatic,hydrophobic, neutral Lys (K) polar, hydrophilic, charged(+) Trp (W)aromatic, hydrophobic, neutral Leu (L) aliphatic, hydrophobic, neutralTyr (Y) aromatic, polar, hydrophobic

TABLE A2 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

The amino acid sequence of a polypeptide of the invention comprises avariant of the amino acid sequence of SEQ ID NO: 2. However, certainresidues in the amino acid sequence of SEQ ID NO: 2 are preferablyretained within the said variant sequence. For example, the said variantsequence typically retains certain residues which are known to berequired for IgG cysteine protease activity. Thus, the cysteine atposition 94 of SEQ ID NO: 1 must be retained (65^(th) residue of SEQ IDNO: 2) in the amino acid sequence of a polypeptide of the invention.Optionally, the lysine at position 84, the histidine at position 262 andthe aspartic acid at each of positions 284 and 286 of SEQ ID NO: 1 arealso retained. These are the 55^(th), 233^(rd), 255^(th) and 257^(th)residues of SEQ ID NO: 2, respectively. Thus, a polypeptide of theinvention typically comprises a variant of the amino acid sequence ofSEQ ID NO: 2 which has a cysteine (C) at the position in said variantsequence which corresponds to position 94 of SEQ ID NO: 1; andoptionally has, at the positions in said variant sequence whichcorrespond to positions 84, 262, 284 and 286 of SEQ ID NO: 1, a lysine(K), a histidine (H), an aspartic acid (D) and an aspartic acid (D),respectively;

Starting with the above structural limitations, the inventors identifiedspecific positions for modification to adjust the functional propertiesof IdeS by assessing a three dimensional model of IdeS. The inventorshave identified the following:

(1) Replacing the asparagine (N) at position 130 of SEQ ID NO: 1 with apositively charged amino acid enhances the potency of a polypeptidewhich incorporates this change. Thus, a polypeptide of the invention maycomprise a variant of the amino acid sequence of SEQ ID NO: 2 which hasa positively charged amino acid at the position in said variant whichcorresponds to position 130 of SEQ ID NO: 1. Common positively chargedamino acids are identified in Table A1 above. The positively chargedamino acid is preferably arginine (R) or lysine (K). Accordingly thisparticular modification may be identified herein by the term “N130R/K”.(2) Replacing the glycine (G) at position 131 of SEQ ID NO: 1 with apositively charged amino acid enhances the potency of a polypeptidewhich incorporates this change. Thus, a polypeptide of the invention maycomprise a variant of the amino acid sequence of SEQ ID NO: 2 which hasa positively charged amino acid at the position in said variant whichcorresponds to position 131 of SEQ ID NO: 1. Common positively chargedamino acids are identified in Table A1 above. The positively chargedamino acid is preferably arginine (R) or lysine (K). Accordingly thisparticular modification may be identified herein by the term “G131R/K”.(3) Deleting the last four residues at the C terminus of SEQ ID NO: 2enhances the potency of a polypeptide which incorporates this change.The last four residues at the C terminus of SEQ ID NO: 2 consist of thecontiguous sequence NQTN. Thus, a polypeptide of the invention maycomprise a variant of the amino acid sequence of SEQ ID NO: 2 which doesnot include the contiguous sequence NQTN. That is, the last fourresidues at the C terminus of SEQ ID NO: 2 may be absent from saidvariant of SEQ ID NO: 2. The last four residues of SEQ ID NO: 2correspond to positions 336-339 of SEQ ID NO: 1. Accordingly thisparticular modification may be identified herein by the term“N336_N339del”.(4) Deleting or altering the sequence of the first twenty residues atthe N terminus of SEQ ID NO: 2 may enhance the potency of a polypeptidewhich incorporates this change and/or may reduce immunogenicity withoutadversely affecting potency.The first twenty residues at the N terminus of SEQ ID NO: 2 consist ofthe contiguous sequence DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 19). Thus, apolypeptide of the invention may comprise a variant of the amino acidsequence of SEQ ID NO: 2 which does not include the contiguous sequenceDSFSANQEIRYSEVTPYHVT.As an example, the said contiguous sequence of SEQ ID NO: 19 may bedeleted in its entirety. That is, the first twenty residues at the Nterminus of SEQ ID NO: 2 may be absent from said variant of SEQ ID NO:2. The first twenty residues of SEQ ID NO: 2 correspond to positions30-49 of SEQ ID NO: 1. Accordingly the deletion of this contiguoussequence may be identified herein by the term “D30_T49del”.Alternatively, a polypeptide of the invention may comprise a variant ofthe amino acid sequence of SEQ ID NO: 2 in which the sequence of thefirst twenty residues at the N terminus of SEQ ID NO: 2 is altered byreplacing one or more amino acids in said first twenty residues. Thealtered sequence preferably has reduced immunogenicity compared to thefirst twenty amino acids of SEQ ID NO: 2. As an example, the amino acidsat positions 2 to 20 of SEQ ID NO: 2 (which corresponding to positions31-49 of SEQ ID NO: 1) may optionally be replaced with the contiguoussequence DYQRNATEAY AKEVPHQIT (SEQ ID NO: 36). In other words, the firsttwenty amino acids of a polypeptide of the invention may consist of thesequence DDYQRNATEA YAKEVPHQIT (SEQ ID NO: 37) instead of the sequenceof SEQ ID NO: 19. The inserted sequence SEQ ID NO: 36 is taken from theN terminal region of IdeZ and corresponds to positions 36-54 of the IdeZNCBI Reference Sequence no WP_014622780.1. Human subjects typically donot express antibodies to IdeZ, and so a polypeptide including thissequence is less prone to ADA. The replacement of SEQ ID NO: 35 with SEQID NO: 37 may be identified herein by the term “S31_T49replZ”.

In summary therefore, a polypeptide of the invention comprises a variantof the sequence of SEQ ID NO: 2, which variant:

-   -   (a) is at least 50% identical to SEQ ID NO: 2;    -   (b) has a cysteine (C) at the position in said variant sequence        which corresponds to position 94 of SEQ ID NO: 1; and optionally    -   (c) has, at the positions in said variant sequence which        correspond to positions 84, 262, 284 and 286 of SEQ ID NO: 1, a        lysine (K), a histidine (H), an aspartic acid (D) and an        aspartic acid (D), respectively;        Preferably, said variant of SEQ ID NO: 2:    -   (1) has a positively charged amino acid at the position in said        variant which corresponds to position 130 of SEQ ID NO: 1,        optionally wherein said positively charged amino acid is        arginine (R) or lysine (K); and/or    -   (2) has a positively charged amino acid at the position in said        variant which corresponds to position 131 of SEQ ID NO: 1,        optionally wherein said positively charged amino acid is        arginine (R) or lysine (K); and/or    -   (3) does not include the contiguous sequence NQTN; and/or    -   (4) does not include the contiguous sequence DSFSANQEIR        YSEVTPYHVT.

Said variant of SEQ ID NO: 2 may include one, two, three or all four ofmodifications (1) to (4) set out above. Said variant may include anycombination of two or three of modifications (1) to (4) set out above. Apreferred variant includes modification (3) and at least one ofmodifications (1) and (2). Another preferred variant includesmodifications (3) and (4) and at least one of modifications (1) and (2).

The inventors have also determined that certain other modifications tothe sequence of SEQ ID NO: 2, which may be applied alternatively or inaddition to any combination of the modifications (1) to (4) describedabove, may increase the potency of a polypeptide of the invention and/ormay reduce the recognition of a polypeptide of the invention byIdeS-specific ADA. Thus, a polypeptide of the invention may comprise avariant of the sequence of SEQ ID NO: 2 in which a substitution is madeat one or more of the positions corresponding to positions 115, 119,139, 142, 198, 216, 226, 241, 245, 302, 316, and 333 of SEQ ID NO: 1.The said variant may comprise a substitution in all of these positions,but typically comprises a substitution in two, three, four, five, six,or seven of these positions. The substitutions in these positionstypically replace the existing amino acid with another amino acid thathas different properties. For example, an uncharged amino acid may bereplaced with a charged amino acid, and vice versa. Preferredsubstitutions at these positions are set out in Table B below using theone letter code:

TABLE B Existing amino acid Position in Preferred in SEQ ID NO: 2 SEQ IDNO: 1 replacement K 115 E E 119 R E 139 K D 142 R E 198 K D 216 N D 226N K 241 E E 245 K S 302 K D 316 K D 333 K

Each of the substitutions in table B may be referred to herein using aterm obtained by combining the entries in the first, second and thirdcolumns for each row from left to right. For example, the substitutionin the first row may be referred to herein as “K115E”, the substitutionin the second row may be referred to as “E119R”, and so on. The specificmodification “D226N” is intended to disrupt a known cell adhesion motifin the sequence of IdeS, that is the contiguous RGD sequence atpositions 224-226 of SEQ ID NO: 1.

Table C below summarizes the modifications made to produce the aminoacid sequences of certain exemplary polypeptides of the invention.

TABLE C Internal Modifications relative to IdeS SEQ ID NO of reference(positions correspond to SEQ ID NO: 1) full sequence pCART152 N130R 3pCART183 N130K, E198R, D216N 4 pCART184 N130R, E198K, D216N, S302K 5pCART185 E119R, D216N, T244D 6 pCART186 E119K, D142R, D216N, T244E,S302K 7 pCART187 K115E, D216N, K241E, E245K, D316K, D333K 8 pCART188E119K, N130R, D142R, D216N, K241S, T244E, E245N, 9 S302K pCART189 E119K,N130R, D142R, E198K, D216N, T244E 10 pCART190 K115E, N130R, E198K,D216N, K241E, E245K, D333K 11 pCART209 N336_N339del (NQTN deletion) 12pCART125 D30_T49del (DSFSANQEIR YSEVTPYHVT deletion) 13 pCART213D30_T49del (DSFSANQEIR YSEVTPYHVT deletion), 14 N130K, D216N PCART214D30_T49del (DSFSANQEIR YSEVTPYHVT deletion), 15 K115E, N130K, E139K,D216N, K241E, E245K, D333K pCART228 S31_T49replZ (replace SEQ ID NO: 35with SEQ ID NO: 36), 16 K115E, N130K, E139K, D216N, K241E, E245K, D333KThe amino acid sequence of SEQ ID NOs: 1 and 2 is reproduced in fullbelow, followed by the amino acid sequence of each of the exemplarypolypeptides of the invention described in Table C.

SEQ ID NO: 1 MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN SEQ ID NO: 2DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART152)SEQ ID NO: 3 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART183)SEQ ID NO: 4 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFKGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKRGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART184)SEQ ID NO: 5 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART185)SEQ ID NO: 6 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLREHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELDEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART186)SEQ ID NO: 7 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLKEHPEKQKINFNGEQMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELEEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART187)SEQ ID NO: 8 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEKNIGAQVLGLFTLS TGQKSWNQTN (pCART188)SEQ ID NO: 9 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLKEHPEKQKINFRGEQMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKSELENGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART189)SEQ ID NO: 10 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLKEHPEKQKINFRGEQMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELEEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSWNQTN (pCART190)SEQ ID NO: 11 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFRGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQKSWNQTN (pCART209)SEQ ID NO: 12 DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQDSW (pCART125)SEQ ID NO: 13 SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (pCART213) SEQ ID NO: 14SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFKGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (pCART214) SEQ ID NO: 15SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFKGEQMFDVKKAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQKSWNQTN (pCART228) SEQ ID NO: 16DDYQRNATEAYAKEVPHQITSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFKGEQMFDVKKAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLS TGQKSWNQTNGGGHHHHHHThe polypeptide of the invention may comprise, consist essentially, orconsist of the sequence of any one of SEQ ID NOs: 3 to 16. Each of SEQID NOs: 3 to 16 may optionally include an additional methionine at the Nterminus and/or a histidine tag at the C terminus. The histidine tag ispreferably consists of six histidine residues. The histidine tag ispreferably linked to the C terminus by a linker of 3× glycine or 5×glycine residues. For SEQ ID NO: 13 the histidine tag is preferablylinked to the C terminus by a linker of 5× glycine residues.

Production of Polypeptides

A polypeptide as disclosed herein may be produced by any suitable means.For example, the polypeptide may be synthesised directly using standardtechniques known in the art, such as Fmoc solid phase chemistry, Bocsolid phase chemistry or by solution phase peptide synthesis.Alternatively, a polypeptide may be produced by transforming a cell,typically a bacterial cell, with a nucleic acid molecule or vector whichencodes said polypeptide. Production of polypeptides by expression inbacterial host cells is described below and is exemplified in theExamples. The invention provides nucleic acid molecules and vectorswhich encode a polypeptide of the invention. The invention also providesa host cell comprising such a nucleic acid or vector. Exemplarypolynucleotide molecules encoding polypeptides disclosed herein areprovided as SEQ ID NOs: 20 to 34. Each of these sequences includes atthe 3′ end a codon for the N terminal methionine (ATG) and, prior to thestop codon (TAA) at the 5′ end, codons for a 3×gly linker and a 6×hishistidine tag, which may optionally be excluded.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include a gene, a genefragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide of theinvention may be provided in isolated or substantially isolated form. Bysubstantially isolated, it is meant that there may be substantial, butnot total, isolation of the polypeptide from any surrounding medium. Thepolynucleotides may be mixed with carriers or diluents which will notinterfere with their intended use and still be regarded as substantiallyisolated. A nucleic acid sequence which “encodes” a selected polypeptideis a nucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences, for example in anexpression vector. The boundaries of the coding sequence are determinedby a start codon at the 5′ (amino) terminus and a translation stop codonat the 3′ (carboxy) terminus. For the purposes of the invention, suchnucleic acid sequences can include, but are not limited to, cDNA fromviral, prokaryotic or eukaryotic mRNA, genomic sequences from viral orprokaryotic DNA or RNA, and even synthetic DNA sequences. Atranscription termination sequence may be located 3′ to the codingsequence.

Polynucleotides can be synthesised according to methods well known inthe art, as described by way of example in Sambrook et al (1989,Molecular Cloning—a laboratory manual; Cold Spring Harbor Press). Thenucleic acid molecules of the present invention may be provided in theform of an expression cassette which includes control sequences operablylinked to the inserted sequence, thus allowing for expression of thepolypeptide of the invention in vivo. These expression cassettes, inturn, are typically provided within vectors (e.g., plasmids orrecombinant viral vectors). Such an expression cassette may beadministered directly to a host subject. Alternatively, a vectorcomprising a polynucleotide of the invention may be administered to ahost subject. Preferably the polynucleotide is prepared and/oradministered using a genetic vector. A suitable vector may be any vectorwhich is capable of carrying a sufficient amount of genetic information,and allowing expression of a polypeptide of the invention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which are positioned in the correct orientation,in order to allow for expression of a peptide of the invention. Othersuitable vectors would be apparent to persons skilled in the art. By wayof further example in this regard we refer to Sambrook et al.

The invention also includes cells that have been modified to express apolypeptide of the invention. Such cells typically include prokaryoticcells such as bacterial cells, for example E. coli. Such cells may becultured using routine methods to produce a polypeptide of theinvention.

A polypeptide may be derivatised or modified to assist with theirproduction, isolation or purification. For example, where a polypeptideof the invention is produced by recombinant expression in a bacterialhost cell, the sequence of the polypeptide may include an additionalmethionine (M) residue at the N terminus to improve expression. Asanother example, the polypeptide of the invention may be derivatised ormodified by addition of a ligand which is capable of binding directlyand specifically to a separation means. Alternatively, the polypeptidemay be derivatised or modified by addition of one member of a bindingpair and the separation means comprises a reagent that is derivatised ormodified by addition of the other member of a binding pair. Any suitablebinding pair can be used. In a preferred embodiment where thepolypeptide for use in the invention is derivatised or modified byaddition of one member of a binding pair, the polypeptide is preferablyhistidine-tagged or biotin-tagged. Typically the amino acid codingsequence of the histidine or biotin tag is included at the gene leveland the polypeptide is expressed recombinantly in E. coli. The histidineor biotin tag is typically present at either end of the polypeptide,preferably at the C-terminus. It may be joined directly to thepolypeptide or joined indirectly by any suitable linker sequence, suchas 3, 4 or 5 glycine residues. The histidine tag typically consists ofsix histidine residues, although it can be longer than this, typicallyup to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2or 1 amino acids.

The amino acid sequence of a polypeptide may be modified to includenon-naturally occurring amino acids, for example to increase stability.When the polypeptides are produced by synthetic means, such amino acidsmay be introduced during production. The polypeptides may also bemodified following either synthetic or recombinant production.Polypeptides may also be produced using D-amino acids. In such cases theamino acids will be linked in reverse sequence in the C to Norientation. This is conventional in the art for producing suchpolypeptides.

A number of side chain modifications are known in the art and may bemade to the side chains of the polypeptides, subject to the polypeptidesretaining any further required activity or characteristic as may bespecified herein. It will also be understood that polypeptides may bechemically modified, e.g. post-translationally modified. For example,they may be glycosylated, phosphorylated or comprise modified amino acidresidues.

The polypeptide may be PEGylated. The polypeptide of the invention maybe in a substantially isolated form. It may be mixed with carriers ordiluents (as discussed below) which will not interfere with the intendeduse and still be regarded as substantially isolated. It may also be in asubstantially purified form, in which case it will generally comprise atleast 90%, e.g. at least 95%, 98% or 99%, of the protein in thepreparation.

Compositions and Formulations Comprising Polypeptides

In another aspect, the present invention provides compositionscomprising a polypeptide of the invention. For example, the inventionprovides a composition comprising one or more polypeptides of theinvention, and at least one pharmaceutically acceptable carrier ordiluent. The carrier (s) must be ‘acceptable’ in the sense of beingcompatible with the other ingredients of the composition and notdeleterious to a subject to which the composition is administered.Typically, carriers and the final composition, are sterile and pyrogenfree.

Formulation of a suitable composition can be carried out using standardpharmaceutical formulation chemistries and methodologies all of whichare readily available to the reasonably skilled artisan. For example,the agent can be combined with one or more pharmaceutically acceptableexcipients or vehicles. Auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, reducing agents and thelike, may be present in the excipient or vehicle. Suitable reducingagents include cysteine, thioglycerol, thioreducin, glutathione and thelike. Excipients, vehicles and auxiliary substances are generallypharmaceutical agents that do not induce an immune response in theindividual receiving the composition, and which may be administeredwithout undue toxicity. Pharmaceutically acceptable excipients include,but are not limited to, liquids such as water, saline,polyethyleneglycol, hyaluronic acid, glycerol, thioglycerol and ethanol.Pharmaceutically acceptable salts can also be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients, vehicles andauxiliary substances is available in Remington's Pharmaceutical Sciences(Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitablefor bolus administration or for continuous administration. Injectablecompositions may be prepared, packaged, or sold in unit dosage form,such as in ampoules or in multi-dose containers containing apreservative. Compositions include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchcompositions may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents. In one embodiment of a composition for parenteraladministration, the active ingredient is provided in dry (for e.g., apowder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration ofthe reconstituted composition. The compositions may be prepared,packaged, or sold in the form of a sterile injectable aqueous or oilysuspension or solution. This suspension or solution may be formulatedaccording to the known art, and may comprise, in addition to the activeingredient, additional ingredients such as the dispersing agents,wetting agents, or suspending agents described herein. Such sterileinjectable formulations may be prepared using a non-toxicparenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides.

Other parentally-administrable compositions which are useful includethose which comprise the active ingredient in microcrystalline form, ina liposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation may comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt. The compositions may be suitable foradministration by any suitable route including, for example,intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial,intraperitoneal, intraarticular, intraosseous or other appropriateadministration routes. Preferred compositions are suitable foradministration by intravenous infusion.

Methods of Use of Polypeptides

The invention provides for the use of polypeptides of the invention invarious methods. For example, the present polypeptides may provideuseful tools for biotechnology. The polypeptides may be used forspecific ex vivo cleavage of IgG, in particular human IgG. In such amethod, the polypeptide may be incubated with a sample containing IgGunder conditions which permit the specific cysteine protease activity tooccur. Specific cleavage can be verified, and the cleavage productsisolated using any suitable method, such as those described inWO2003051914 and WO2009033670. Thus the method can be used in particularto generate Fc and F(ab′)₂ fragments. Fab fragments may then be producedby carrying out a reduction step (for example in 2-mercaptoethanolamineor Cysteamine) on the F(ab′)₂ fragments that result from cleavage of IgGwith a polypeptide of the invention.

The method may also be used to detect or analyse IgG in a sample, or toremove IgG from a sample. A method for the detection of IgG in a sampletypically involves incubating the polypeptide with the sample underconditions which permit IgG-specific binding and cleavage. The presenceof IgG can be verified by detection of the specific IgG cleavageproducts, which may subsequently be analysed.

The polypeptides in accordance with the present invention may also beused in therapy or prophylaxis. In therapeutic applications,polypeptides or compositions are administered to a subject alreadysuffering from a disorder or condition, in an amount sufficient to cure,alleviate or partially arrest the condition or one or more of itssymptoms. Such therapeutic treatment may result in a decrease inseverity of disease symptoms, or an increase in frequency or duration ofsymptom-free periods. An amount adequate to accomplish this is definedas “therapeutically effective amount”. In prophylactic applications,polypeptides or compositions are administered to a subject not yetexhibiting symptoms of a disorder or condition, in an amount sufficientto prevent or delay the development of symptoms. Such an amount isdefined as a “prophylactically effective amount”. The subject may havebeen identified as being at risk of developing the disease or conditionby any suitable means. Thus the invention also provides a polypeptide ofthe invention for use in the treatment of the human or animal body. Alsoprovided herein is a method of prevention or treatment of disease orcondition in a subject, which method comprises administering apolypeptide of the invention to the subject in a prophylactically ortherapeutically effective amount. The polypeptide may be co-administeredwith an immune-suppressive agent. The polypeptide is preferablyadministered by intravenous infusion, but may be administered by anysuitable route including, for example, intradermal, subcutaneous,percutaneous, intramuscular, intra-arterial, intraperitoneal,intraarticular, intraosseous or other appropriate administration routes.The amount of said polypeptide that is administered may have a lowerlimit of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21,0.22, 0.23, 0.24, 0.25 mg/kg BW. The amount of said polypeptide that isadministered may have an upper limit of about 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg/kgBW. It would be understood that any of the above mentioned upper limitsmay be combined with any of the above lower limits to provide a rangefor the dose that is administered. For example, the amount of saidpolypeptide that is administered may be between about 0.01 mg/kg BW and2 mg/kg BW, between 0.10 and 1 mg/kg BW, and preferably between 0.25mg/kg BW and 0.5 mg/kg BW. The polypeptide may be administered onmultiple occasions to the same subject, provided that the quantity ofADA in the serum of the subject which is capable of binding to thepolypeptide does not exceed a threshold determined by the clinician. Thequantity of ADA in the serum of the subject which is capable of bindingto the polypeptide may be determined by any suitable method, such as anagent specific CAP 1-BIA (ImmunoCAP) test or a titre assay.

Polypeptides of the invention may be particularly useful in thetreatment or prevention of a disease or condition mediated by pathogenicIgG antibodies. Accordingly, the invention provides a polypeptide of theinvention for use in the treatment or prevention of a disease orcondition mediated by pathogenic IgG antibodies. The invention alsoprovides a method of treating or preventing a disease or conditionmediated by pathogenic IgG antibodies comprising administering to anindividual a polypeptide of the invention. The method may compriserepeat administration of the said polypeptide. The invention alsoprovides a polypeptide of the invention for use in the manufacture of amedicament for the treatment or prevention of a disease or conditionmediated by pathogenic IgG antibodies.

The pathogenic antibodies may typically be specific for an antigen whichis targeted in an autoimmune disease or other condition mediated whollyor in part by antibodies. Table D sets out a list of such diseases andthe associated antigens. A polypeptide of the invention may be used totreat any of these diseases or conditions. The polypeptide isparticularly effective for the treatment or prevention of autoimmunedisease which is mediated in whole or in part by pathogenic IgGantibodies.

TABLE D DISEASE AUTOANTIGENS Addison's disease Steroid 21-hydroxylase,17 alpha-Hydroxylase (17OH) and side-chain-cleavage enzyme (P450scc),Thyroperoxidase, thyroglobulin and H+/K(+)− Anti-GBM glomerulonephritisAnti-glomerular basement membrane (anti-GBM): (related to Goodpasteur)noncollagenous (NC1) domains of the alpha3alpha4alpha5(IV) collagenAnti-neutrophil cytoplasmic Myeloperoxidase, proteinase 3antibody-associated vasculitides (ANCA associated vasculitis)(Wegenergranulomatosis, Churg-Strauss syndrome, microscopic polyangiitis)Anti-NMDAR Encephalitis N-methyl-D-aspartate receptor (NMDAR)Anti-phospholipid antibody Negatively-charged phospholipids complexedwith syndrome (APS) and catastrophic phospholipid binding plasmaproteins (e.g. beta2GPI), APS cardiolipin, beta2-glycoprotein I, and(beta2GPI) Autoimmune bullous skin diseases IgG against keratinocytes.Specific target is desmoglein (Dsg) (Pemphigus). Pemphigus foliaceus 1(desmosomal (PF), fogo selvagem (FS)(endemic Cadherins) form), pemphigusvulgaris (PV) Autoimmune hemolytic anemia Self-antigens onred-blood-cells (AIHA) Autoimmune hepatitis (AIH) Actin, antinuclearantibody (ANA), smooth muscle antibody (SMA), liver/kidney microsomalantibody (LKM-1), anti soluble liver antigen (SLA/LP) andanti-mitochondrial antibody (AMA), CYP2D6, CYP2C9-tienilic acid, UGT1A,CYP1A2, CYP2A6, CYP3A, CYP2E1, CYP11A1, CYP17 and CYP21 Autoimmuneneutropenia (AIN) FcgRIIIb Bullous pemphigoid (BP) Hemidesmosomalproteins BP230 and BP180 (type XVII collagen), laminin 5, the alpha6subunit of the integrin alpha6beta4 and p200 Celiac diseasetransglutaminase 2 (TG2), transglutaminase 3, actin, ganglioside,collagen, calreticulin and zonulin, thyroid, endocrine pancreas,anti-gastric and liver, anti-nuclear constituents, anti-reticulin,actin, smooth muscle, calreticulin, desmin, collagens, bone, anti-brain,ganglioside, neuronal, blood vessel Chronic utricaria Alpha-subunit ofthe high-affinity IgE receptor, IgE Complete congenital heart block Ro(Sjögens syndrome antigen A (SSA)), La (Sjögens syndrome (CCHB) antigenB(SSB)) Diabetes type 1A (T1DM) Islet cell autoantibodies (ICA),antibodies to insulin (IAA), glutamic acid decarboxylase (GAA or GAD),protein tyrosine phosphatase (IA2 or ICA512), Insulinoma AssociatedPeptide- 2. The number of antibodies, rather than the individualantibody, is thought to be most predictive of progression to overtdiabetes. Epidermolysis bullosa acquisita The 145-kDa noncollagenousaminoterminal (NC-1) domain of (EBA) collagen VII Essential mixedcryoglobulinemia Essential mixed cryoglobulinemia antigens Goodpasture'ssyndrome (also known alpha3(IV) collagen (=Goodpasture antigen) asGoodpasture's disease and anti-glomerular basement membrane diseaseGraves'disease (Basedow's disease), Thyrotropin receptor (TSHR) Thyroidperoxidase (TPO) includes Goitre and hyperthyroidism, infiltrativeexopthalmos and infiltarative dermopathy. Guillain-Barré syndrome (GBS).Gangliosides GM1, GM1b, GD1a, and GalNAc-GD1a, Acute inflammatorydemyelinating glycosphingolipid, myelin proteins PMP22 and P0polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN) Hemophilia -Acquired FVIII Factor VIII deficiency Idiopathic thrombocytopenicpurpura Platelet glycoprotein (GP) IIb-IIIa and/or GPIb-IX (ITP)Lambert-Eaton myasthenic syndrome voltage gated calcium channels (LEMS)Mixed Connective Tissue Disease IgG directed against the spliceosome,U1-snRNP (MCTD) Multiple Myeloma Multiple Myeloma antigens Myastheniagravis Acetylcholine receptors (AchR), muscle-specific kinase Myastheniccrisis (MuSK) Myocarditis, dilated cardiomyopathy heart-reactiveautoantibodies against multiple antigens e.g. (DCM)(congestivecardiomyopathy) cardiac myosin Neuromyelitis Optica (NMO) Aquaporin 4(AQP4) Primary biliary cirrhosis (PBC) pyruvate dehydrogenase complex(PDC)-E2 and other members of the oxaloacid dehydrogenase family,Glycoprotein-210, p62, sp100 Primary Progressive Multiple Myelinoligodendrocyte glycoprotein (MOG), Myelin Sclerosis (PPMS) proteolipidprotein (PLP), transketolase (TK), cyclic nucleotide phosphodiesterasetype I (CNPase I), collapsin response mediator protein 2, tubulin beta4,neurofascin Rheumatic heart disease Cardiac myosin (RHD), (Rheumaticfever) Rheumatoid Arthritis (RA) Type II collagen, citrullin (-atedproteins (e.g. (fibrinogen, vimentin, filaggrin, type II collagen,enolase)), G6PI, RFs (anti- Fc/IgG), Vimentin, and cytokeratinSerum-sickness, immune complex Various antigens hypersensitivity (typeIII) Sjogren Syndrome (SS) Ro (Sjögens syndrome antigen A (SS-A)), La(Sjögens syndrome antigen B(SS-B)), p80 coilin, antinuclear antibodies,anti-thyroid, anti-centromere antibodies (Raynaud's phenomenon),anti-carbonic anhydrase II (distal renal tubular acidosis),anti-mitochondrial antibodies (liver pathology), cryoglobulins(evolution to non-Hodgkin's lymphoma), alpha- and beta-fodrin, isletcell autoantigen, poly(ADP)ribose polymerase (PARP), NuMA, Golgins, NOR-90, M3-muscarinic receptor SLE including Lupus nephritis Autoantibodiesto nuclear constituents (e.g. dsDNA and nucleosomes), dsDNA, PARP, Sm,PCDA, rRNA Ribosome P proteins, C1q Stiff-person syndrome (SPS) glutamicacid decarboxylase (GAD), amphiphysin. Systemic sclerosis (scleroderma)DNA-topoisomerase I (Scl-70), U3 snRNP, U2 snRNP, 7-2 RNP, NOR-90,centromere-associated proteins, and nucleolar antigens, Anti-Th/To,Anti-RNA polymerase I/III, Anti-PDGF receptor, Anti-fibrillin-1, M3-muscarinic receptor, Transplant rejection Transplant rejection antigensThrombotic Thrombocytopenic ADAMTS13 Purpura (TTP)

In another embodiment, a polypeptide of the invention may be used in amethod to improve the benefit to a subject of a therapy or a therapeuticagent. The method comprises two steps, which are referred to herein assteps (a) and (b).

Step (a) comprises administering to the subject a polypeptide of theinvention. The amount of the polypeptide administered is preferablysufficient to cleave substantially all IgG molecules present in theplasma of the subject. Step (b) comprises subsequently administering tothe subject the said therapy or therapeutic agent. Steps (a) and (b) areseparated by a time interval which is preferably sufficient for cleavageof substantially all IgG molecules present in the plasma of the subjectto take place. The said interval may typically be of at least 30 minutesand at most 21 days.

The therapeutic agent of which the benefit is improved is typically anantibody which is administered for the treatment of cancer or anotherdisease. The therapeutic agent may be IVIg. In the context of thisembodiment, the invention may be alternatively described as providing amethod for the treatment of cancer or another disease in a subject, themethod comprising (a) administering to the subject a polypeptide of theinvention; and (b) subsequently administering to the subject atherapeutically effective amount of an antibody which is a treatment forsaid cancer or said other disease; wherein:

-   -   the amount of said polypeptide administered is sufficient to        cleave substantially all IgG molecules present in the plasma of        the subject; and    -   steps (a) and (b) are separated by a time interval of at least 2        hours and at most 21 days.

In other words, the invention also provides the polypeptide for use insuch a method for the treatment of cancer or another disease. Theinvention also provides use of the agent in the manufacture of amedicament for the treatment of cancer or another disease by such amethod. The cancer may be Acute lymphoblastic leukemia, Acute myeloidleukemia, Adrenocortical carcinoma, AIDS-related cancers, AIDS-relatedlymphoma, Anal cancer, Appendix cancer, Astrocytoma, childhoodcerebellar or cerebral, Basal cell carcinoma, Bile duct cancer,extrahepatic, Bladder cancer, Bone cancer, Osteosarcoma/Malignantfibrous histiocytoma, Brainstem glioma, Brain cancer, Brain tumor,cerebellar astrocytoma, Brain tumor, cerebral astrocytoma/malignantglioma, Brain tumor, ependymoma, Brain tumor, medulloblastoma, Braintumor, supratentorial primitive neuroectodermal tumors, Brain tumor,visual pathway and hypothalamic glioma, Breast cancer, Bronchialadenomas/carcinoids, Burkitt lymphoma, Carcinoid tumor, Carcinoid tumor,gastrointestinal, Carcinoma of unknown primary, Central nervous systemlymphoma, Cerebellar astrocytoma, Cerebral astrocytoma/Malignant glioma,Cervical cancer, Chronic lymphocytic leukemia, Chronic myelogenousleukemia Chronic myeloproliferative disorders, Colon Cancer, CutaneousT-cell lymphoma, Desmoplastic small round cell tumor, Endometrialcancer, Ependymoma, Esophageal cancer, Ewing's sarcoma in the Ewingfamily of tumors, Extracranial germ cell tumor, Childhood, ExtragonadalGerm cell tumor, Extrahepatic bile duct cancer, Eye Cancer, Intraocularmelanoma, Eye Cancer, Retinoblastoma, Gallbladder cancer, Gastric(Stomach) cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinalstromal tumor (GIST), Germ cell tumor: extracranial, extragonadal, orovarian, Gestational trophoblastic tumor, Glioma of the brain stem,Glioma, Childhood Cerebral Astrocytoma, Glioma, Childhood Visual Pathwayand Hypothalamic, Gastric carcinoid, Hairy cell leukemia, Head and neckcancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma,Hypopharyngeal cancer, Hypothalamic and visual pathway glioma,Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposisarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias,Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia),Leukemia, acute myeloid (also called acute myelogenous leukemia),Leukemia, chronic lymphocytic (also called chronic lymphocyticleukemia), Leukemia, chronic myelogenous (also called chronic myeloidleukemia), Leukemia, hairy cell, Lip and Oral Cavity Cancer,Liposarcoma, Liver Cancer (Primary), Lung Cancer, Non-Small Cell LungCancer, Small Cell, Lymphomas, Lymphoma, AIDS-related, Lymphoma,Burkitt, Lymphoma, cutaneous T-Cell, Lymphoma, Hodgkin, Lymphomas,Non-Hodgkin (an old classification of all lymphomas except Hodgkin's),Lymphoma, Primary Central Nervous System, Macroglobulinemia,Waldenström, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma,Medulloblastoma, Melanoma, Melanoma, Intraocular (Eye), Merkel CellCarcinoma, Mesothelioma, Adult Malignant, Mesothelioma, MetastaticSquamous Neck Cancer with Occult Primary, Mouth Cancer, MultipleEndocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm,Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia,Chronic, Myeloid Leukemia, Adult Acute, Myeloid Leukemia, ChildhoodAcute, Myeloma, Multiple (Cancer of the Bone-Marrow), MyeloproliferativeDisorders, Nasal cavity and paranasal sinus cancer, Nasopharyngealcarcinoma, Neuroblastoma, Non-Hodgkin lymphoma, Non-small cell lungcancer, Oral Cancer, Oropharyngeal cancer, Osteosarcoma/malignantfibrous histiocytoma of bone, Ovarian cancer, Ovarian epithelial cancer(Surface epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian lowmalignant potential tumor, Pancreatic cancer, Pancreatic cancer, isletcell, Paranasal sinus and nasal cavity cancer, Parathyroid cancer,Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma,Pineal germinoma, Pineoblastoma and supratentorial primitiveneuroectodermal tumors, Pituitary adenoma, Plasma cellneoplasia/Multiple myeloma, Pleuropulmonary blastoma, Primary centralnervous system lymphoma, Prostate cancer, Rectal cancer, Renal cellcarcinoma (kidney cancer), Renal pelvis and ureter, transitional cellcancer, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer,Sarcoma, Ewing family of tumors, Kaposi Sarcoma, Sarcoma, soft tissue,Sarcoma, uterine, Sézary syndrome, Skin cancer (nonmelanoma), Skincancer (melanoma), Skin carcinoma, Merkel cell, Small cell lung cancer,Small intestine cancer, Soft tissue sarcoma, Squamous cell carcinoma,Squamous neck cancer with occult primary, metastatic, Stomach cancer,Supratentorial primitive neuroectodermal tumor, T-Cell lymphoma,cutaneous—see Mycosis Fungoides and Sézary syndrome, Testicular cancer,Throat cancer, Thymoma, Thymoma and Thymic carcinoma, Thyroid cancer,Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter,Trophoblastic tumor, Ureter and renal pelvis, transitional cell cancerUrethral cancer, Uterine cancer, endometrial, Uterine sarcoma, Vaginalcancer, Visual pathway and hypothalamic glioma, Vulvar cancer,Waldenström macroglobulinemia and Wilms tumor (kidney cancer).

The cancer is preferably prostate cancer, breast cancer, bladder cancer,colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, lungcancer, cervical cancer, endometrial cancer, kidney (renal cell) cancer,oesophageal cancer, thyroid cancer, skin cancer, lymphoma, melanoma orleukemia.

The antibody administered in step (b) is preferably specific for atumour antigen associated with one or more of the above cancer types.Targets of interest for an antibody for use in the method include CD2,CD3, CD19, CD20, CD22, CD25, CD30, CD32, CD33, CD40, CD52, CD54, CD56,CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD138, CD174, CD205, CD227,CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphA2, EphB2,FAP, av integrin, Mesothelin, EGFR, TAG-72, GD2, CA1X, 5T4, α4β7integrin, Her2. Other targets are cytokines, such as interleukins IL-Ithrough IL-13, tumour necrosis factors α and β, interferons α, β and γ,tumour growth factor Beta (TGF-β), colony stimulating factor (CSF) andgranulocyte monocyte colony stimulating factor (GMCSF). See HumanCytokines: Handbook for Basic & Clinical Research (Aggrawal et al. eds.,Blackwell Scientific, Boston, Mass. 1991). Other targets are hormones,enzymes, and intracellular and intercellular messengers, such as, adenylcyclase, guanyl cyclase, and phospholipase C. Other targets of interestare leukocyte antigens, such as CD20, and CD33. Drugs may also betargets of interest. Target molecules can be human, mammalian orbacterial. Other targets are antigens, such as proteins, glycoproteinsand carbohydrates from microbial pathogens, both viral and bacterial,and tumors. Still other targets are described in U.S. Pat. No.4,366,241.

The antibody may be attached directly or indirectly to a cytotoxicmoiety or to a detectable label. The antibody may be administered viaone or more routes of administration using one or more of a variety ofmethods known in the art. The route and/or mode of administration willvary depending upon the desired results. Preferred routes ofadministration for antibodies include intravenous, intramuscular,intradermal, intraperitoneal, subcutaneous, spinal or other parenteralroutes of administration, for example by injection or infusion. Thephrase “parenteral administration” as used herein means modes ofadministration other than enteral and topical administration, usually byinjection. Alternatively, an antibody can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration. Local administration is also preferred, includingperitumoral, juxtatumoral, intratumoral, intralesional, perilesional,intra cavity infusion, intravesicle administration, and inhalation.

A suitable dosage of an antibody of the invention may be determined by askilled medical practitioner. Actual dosage levels of an antibody may bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularantibody employed, the route of administration, the time ofadministration, the rate of excretion of the antibody, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A suitable dose of an antibody may be, for example, in the range of fromabout 0.1 μg/kg to about 100 mg/kg body weight of the patient to betreated. For example, a suitable dosage may be from about 1 μg/kg toabout 10 mg/kg body weight per day or from about 10 μg/kg to about 5mg/kg body weight per day.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, or step (b) of the method may comprise several divideddoses administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation, provided the required interval between steps (a) and (b) isnot exceeded. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

The antibody of step (b) may be administered in combination withchemotherapy or radiation therapy. The method may further comprises theadministration of an additional anti-cancer antibody or othertherapeutic agent, which may be administered together with the antbodyof step (b) in a single composition or in separate compositions as partof a combined therapy. For example, the antibody of step (b) may beadministered before, after or concurrently with the other agent.

The antibody may be Abagovomab, Abciximab, Actoxumab, Adalimumab,Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518,Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomabmafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab,Atlizumab (=tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab,Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab,Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab,Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin,Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumabravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab,CC49, Cedelizumab, Certolizumab pegol, Cetuximab, Ch.14.18, Citatuzumabbogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumabtetraxetan, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab,Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab,Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab,Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab,Elotuzumab Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab,Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab,Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab,Fanolesomab, Faralimomab Farletuzumab, Fasinumab, FBTA05, Felvizumab,Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab,Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab,Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin,Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab,Gomiliximab, GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab,Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine,Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab,Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab,Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab,Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab,Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab,Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab,Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab,Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox,Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab,Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab,Nofetumomab merpentan, Obinutuzumab, Ocaratuzumab, Ocrelizumab,Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab,Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab,Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab,Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab,Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab,Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin,Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab,Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab,Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab,Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab,Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab,Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab,Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab,Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab,Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomabpaptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab,Teplizumab, Teprotumumab, TGN1412, Ticilimumab (=tremelimumab),Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (=atlizumab),Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07,Tregalizumab, Tremelimumab Tucotuzumab celmoleukin, Tuvirumab,Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab,Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab Vesencumab,Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab,Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab or Zolimomab aritox.

Preferred antibodies include Natalizumab, Vedolizumab, Belimumab,Atacicept, Alefacept, Otelixizumab, Teplizumab, Rituximab, Ofatumumab,Ocrelizumab, Epratuzumab, Alemtuzumab, Abatacept, Eculizamab,Omalizumab, Canakinumab, Meplizumab, Reslizumab, Tocilizumab,Ustekinumab, Briakinumab, Etanercept, Inlfliximab, Adalimumab,Certolizumab pegol, Golimumab, Trastuzumab, Gemtuzumab, Ozogamicin,Ibritumomab, Tiuxetan, Tostitumomab, Cetuximab, Bevacizumab,Panitumumab, Denosumab, Ipilimumab, Brentuximab and Vedotin.

The therapy of which the benefit is improved is typically an organtransplant. The organ may be selected from kidney, liver, heart,pancreas, lung, or small intestine. The subject to be treated maypreferably be sensitized or highly sensitized. By “sensitized” it ismeant that the subject has developed antibodies to human majorhistocompatibility (MHC) antigens (also referred to as human leukocyteantigens (HLA)). The anti-HLA antibodies originate from allogenicallysensitized B-cells and are usually present in patients that havepreviously been sensitized by blood transfusion, previoustransplantation or pregnancy (Jordan et al., 2003).

Whether or not a potential transplant recipient is sensitized may bedetermined by any suitable method. For example, a Panel ReactiveAntibody (PRA) test may be used to determine if a recipient issensitized. A PRA score >30% is typically taken to mean that the patientis “high immunologic risk” or “sensitized”. Alternatively, a cross matchtest may be conducted, in which a sample of the potential transplantdonor's blood is mixed with that of the intended recipient. A positivecross-match means that the recipient has antibodies which react to thedonor sample, indicating that the recipient is sensitized andtransplantation should not occur. Cross-match tests are typicallyconducted as a final check immediately prior to transplantation.

The presence of high titer antibodies against MHC antigens of thepotential donor (i.e. donor specific antibodies (DSA)) is a directcontraindication to transplantation because of the risk of acuteantibody-mediated rejection. In short, sensitization to donor MHCantigens hampers the identification of a suitable donor. A positivecross-match test is an unambiguous barrier to transplantation. Sinceapproximately one third of patients waiting for kidney transplantationare sensitized, with as many as 15% being highly sensitized, this leadsto an accumulation of patients waiting for transplant. In the US, themedian time on the waiting list for renal transplantation in 2001-2002was 1329 days for those with Panel Reactive Antibody (PRA) score 0-9%,1920 days for those with PRA 10-79%, and 3649 days for those with PRA80% or greater (OPTN-database, 2011).

One accepted strategy to overcome the DSA barrier is to apply plasmaexchange or immune adsorption, often in combination with e.g.intravenous gamma globulin (IVIg) or Rituximab, to lower the levels ofDSA to a level where transplantation can be considered (Jordan et al.,2004; Montgomery et al., 2000; Vo et al., 2008a; Vo et al., 2008b).However, plasma exchange, immune adsorption and IVIg treatments have thedisadvantage of being inefficient and requiring rigorous planning sincethey involve repeated treatments over an extended period of time. Whenan organ from a deceased donor becomes available it has to betransplanted within hours since prolonged cold ischemia time is one ofthe most important risk factors for delayed graft function and allograftloss in renal transplantation (Ojo et al., 1997).

By contrast, the method of the present invention allows the rapid,temporary and safe removal of DSAs in a potential transplant recipient.Administering the polypeptide of the invention just prior totransplantation has the capacity to effectively desensitize a highlysensitized patient, thereby allowing transplantation and avoiding acuteantibody-mediated rejection. A single dose of polypeptide prior totransplantation will enable transplantation of thousands of patientswith donor specific IgG antibodies.

In the context of this embodiment, the method may be alternativelydescribed as a method for the treatment of organ failure in a subject,the method comprising (a) administering to the subject a polypeptide ofthe invention and (b) subsequently transplanting a replacement organinto the subject; wherein:

-   -   the amount of said polypeptide administered is sufficient to        cleave substantially all IgG molecules present in the plasma of        the subject; and    -   steps (a) and (b) are separated by a time interval of at least 2        hours and at most 21 days.

In other words, this embodiment may be described as a method forpreventing rejection of a transplanted organ in a subject, particularlyacute antibody-mediated transplant rejection, the method comprising, atleast 2 hours and at most 21 days prior to transplantation of the organ,administering to the subject a polypeptide of the invention, wherein theamount of said polypeptide administered is sufficient to cleavesubstantially all IgG molecules present in the plasma of the subject.The invention also provides use of the polypeptide of the invention insuch a method of treating organ failure or preventing transplantrejection, particularly acute antibody-mediated transplant rejection.The invention also provides use of the polypeptide of the invention inthe manufacture of a medicament for the treatment of organ failure orfor the prevention of transplant rejection by such a method. In thisembodiment, the method of the invention may additionally comprise a stepconducted at or immediately prior to transplantation, which stepcomprises induction suppression of T cells and/or B cells in thepatient. Said induction suppression may typically comprise administeringan effective amount of an agent which kills or inhibits T cells, and/oradministering an effective amount of an agent which kills or inhibits Bcells. Agents which kill or inhibit T cells include Muromonab,Basiliximab, Daclizumab, an antithymocyte globulin (ATG) antibody and alymphocyte immune globulin, anti-thymocyte globulin preparation (ATGAM).Rituximab is known to kill or inhibit B cells.

EXAMPLES

Unless indicated otherwise, the methods used are standard biochemistryand molecular biology techniques. Examples of suitable methodologytextbooks include Sambrook et al., Molecular Cloning, A LaboratoryManual (1989) and Ausubel et al., Current Protocols in Molecular Biology(1995), John Wiley and Sons, Inc.

Example 1—Design of Polypeptides, Production and Purification

The mature IdeS molecule was analysed and regions suitable for mutationwere identified. In some cases an in silico assessment was used toevaluate the likely outcome of a mutation. Having decided on thesequence of each polypeptide, cDNA encoding each polypeptide weregenerated at GeneCust, Luxembourg either by site-directed mutation of astarting sequence or synthesis depending on the number of mutationsintroduced. cDNA were sequenced and transferred to the pET9a expressionvector (Novagene) in frame with a C-terminal 6×His-tag, joined to theC-terminus by a short glycine linker (3×Gly). N terminal methionine wasadded to improve bacterial expression. The plasmids were transformed(heat-shock) into E. coli BL21(DE3) (Stratagene) and seeded on LBagarose plates containing 30 μg/ml kanamycin. Single colonies werepicked and overnight cultures (3 ml LB-medium) were started at 37° C.,250 rpm. The following day glycerol stocks were prepared and 10 mlTB-medium supplemented with 30 μg/ml kanamycin and anti-foam wereinoculated with overnight culture and grown until OD 0.6-0.8 (37° C.,300 rpm). At this point IPTG (1 mM) was added and cultures werecontinued for 1 hour prior to harvest of the bacteria by centrifugation.The pellets were washed in PBS and frozen at −20° C. A freeze-thawprotocol for bacterial lysis was used (three freeze/thaw cycles in 1 mlPBS each) and the proteins were purified using Ni-NTA pre-packedspin-columns (Pierce). After purification the eluted proteins wereactivated with 10 mM DTT prior to buffer exchange (3 volumes PBS in MWCO9K Millipore cfg devises). The purity and stability of each protein wasevaluated using sodium dodecyl sulphate polyacrylamide gelelectrophoreses (SDS-PAGE) stainless 12% Mini-PROTEAN®TGX™ precast gel(Biorad) SDS-PAGE

The following table summarises the changes made for each testedpolypeptide relative to mature IdeS, not including the additional Nterminal methionine and his tag. Thus, the sequence of each polypeptideused in the experiments described herein typically comprises thesequence of the SEQ ID NO as indicated in the table, plus an additionalN terminal methionine and a his tag joined to the C terminal end by ashort glycine linker.

Internal Modifications relative to IdeS reference (positions correspondto SEQ ID NO: 1) SEQ ID NO pCART152 N130R 3 pCART183 N130K, E198R, D216N4 pCART184 N130R, E198K, D216N, S302K 5 pCART185 E119R, D216N, T244D 6pCART186 E119K, D142R, D216N, T244E, S302K 7 pCART187 K115E, D216N,K241E, E245K, D316K, D333K 8 pCART188 E119K, N130R, D142R, D216N, K241S,T244E, E245N, 9 S302K pCART189 E119K, N130R, D142R, E198K, D216N, T244E10 pCART190 K115E, N130R, E198K, D216N, K241E, E245K, D333K 11 pCART209N336_N339del (NQTN deletion) 12 pCART125 D30_T49del (DSFSANQEIRYSEVTPYHVT deletion) 13 pCART213 D30_T49del (DSFSANQEIR YSEVTPYHVTdeletion), 14 N130K, D216N pCART214 D30_T49del (DSFSANQEIR YSEVTPYHVTdeletion), 15 K115E, N130K, E139K, D216N, K241E, E245K, D333K pCART228S31_T49replZ (replace SEQ ID NO: 35 with SEQ ID NO: 36), 16 K115E,N130K, E139K, D216N, K241E, E245K, D333K

As a control, a version of IdeS was produced using the same methodologyas described above. This version of IdeS comprises the sequence of SEQID NO: 2 plus an additional N terminal methionine and a his tag joinedto the C terminal end by a short glycine linker. This version of IdeSmay be referred to herein as pCART124. The sequence of pCART124 isprovided below:

(SEQ ID NO: 17) MDSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTL STGQDSWNQTNGGGHHHHHH

IdeS lacking tag was also independently produced to GMP standard usingautomated multistep chromatographic purification, for use as a furthercontrol. This polypeptide is referred to herein as BX1001865.

The cDNA sequence used to produce each of the tested polypeptides andpCART124 is provided below. Each cDNA sequence includes at the 3′ end acodon for the N terminal methionine (ATG) and, prior to the stop codon(TAA) at the 5′ end, codons for the glycine linker and the histidinetag.

pCART124 (IdeS; SEQ ID NO: 20)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAATGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTGATCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART152 (SEQ ID NO: 21)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCCGTGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTGATCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART183 (SEQ ID NO: 22)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAAAGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAACGTGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART184 (SEQ ID NO: 23)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCCGTGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAAAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATAAAGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART185 (SEQ ID NO: 24)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGCGTGAGCATCCAGAAAAGCAAAAAATAAACTTCAATGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAGATGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART186 (SEQ ID NO: 25)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGAAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAATGGCGAACAGATGTTTGACGTAAAAGAAGCTATCCGTACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAGAAGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATAAAGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART187 (SEQ ID NO: 26)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTGAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAATGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGGAAGAGTTAACCAAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAAAAAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAAAAAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART188 (SEQ ID NO: 27)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGAAAGAGCATCCAGAAAAGCAAAAAATAAACTTCCGTGGCGAACAGATGTTTGACGTAAAAGAAGCTATCCGTACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAGTGAGTTAGAAAACGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATAAAGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART189 (SEQ ID NO: 28)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGAAAGAGCATCCAGAAAAGCAAAAAATAAACTTCCGTGGCGAACAGATGTTTGACGTAAAAGAAGCTATCCGTACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAAAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAGAAGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART190 (SEQ ID NO: 29)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTGAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCCGTGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAAAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGGAAGAGTTAACCAAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAAAAAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAApCART209 (SEQ ID NO: 30)ATGGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCACGTTACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAATGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTGATCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGGGTGGCGGCGGTGGCCATCATCACCATCA CCACTAA pCART125(SEQ ID NO: 31) ATGTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAATGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTGATCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCACCATCACCAC TAA pCART213(SEQ ID NO: 32) ATGTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTAAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAAAGGCGAACAGATGTTTGACGTAAAAGAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGTTGGAATCAGACCAATGGCGGTGGCCATCATCACCATCACCAC TAA pCART214(SEQ ID NO: 33) ATGTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTGAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAAAGGCGAACAGATGTTTGACGTAAAAAAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGGAAGAGTTAACCAAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAAAAAGTTGGAATCAGACCAATGGCGGTGGCCATCATCACCATCACCAC TAA pCART228(SEQ ID NO: 34) ATGGACGATTACCAAAGGAATGCTACGGAAGCTTATGCCAAAGAAGTACCACATCAGATCACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAACTTCACTCAAGGTGAAGATGTTTTTCACGCTCCTTATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCTGCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGACCAAATTGAACGTTATTTGGAAGAGCATCCAGAAAAGCAAAAAATAAACTTCAAAGGCGAACAGATGTTTGACGTAAAAAAAGCTATCGACACTAAAAACCACCAGCTAGATAGTAAATTATTTGAATATTTTAAAGAAAAAGCTTTCCCTTATCTATCTACTAAACACCTAGGAGTTTTCCCTGATCATGTAATTGATATGTTCATTAACGGCTACCGCCTTAGTCTAACTAACCACGGTCCAACGCCAGTAAAAGAAGGTAGTAAAGATCCCCGAGGTGGTATTTTTGACGCCGTATTTACAAGAGGTAACCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTCATTAAGGAAGAGTTAACCAAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCATGTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGATAGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCTAAAGAAATAAAAGAAGATAATATAGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAAAAAGTTGGAATCAGACCAATGGCGGTGGCCATCATCA CCATCACCACTAA

Example 2—Assessment of Potency (IgG Cleavage Efficacy) ELISA

Enzymatic activity was measured using an ELISA-based potency assay. Theprinciple of the ELISA was to coat wells of a multi titre plate with anantibody target (BSA), then incubate different concentrations of IgGcysteine protease polypeptide (test or control) with anti-BSA antibodyin the wells, before detecting the quantity of anti-BSA antibody boundto the wells using a detector antibody. The higher the concentration ofa given IgG cysteine protease polypeptide in a well, the less intactanti-BSA polypeptide will be bound to the well, giving a lower signal.Similarly, a more potent IgG cysteine protease polypeptide will give alower signal than a less potent IgG cysteine protease polypeptide whenpresent at the same concentration.

The reference IdeS BX1001865 was prepared as a titration series in 1:2dilution steps from 320 nM down to 0.16 nM to allow plotting of astandard calibration curve for the assay. The results achieved in theassay for multiple known concentrations of each tested polypeptide werecompared against the linear section of the calibration curve todetermine the concentration of reference IdeS which achieved the samepotency. Dividing the known concentration of each polypeptide by thedetermined equivalent concentration of reference IdeS from the curve, ascore is produced which is the fold change in potency relative toreference IdeS BX1001865. For example, if 5 nM test polypeptide achievesa result equivalent to 10 nM reference IdeS on the calibration curve,the test polypeptide has a potency 2 fold greater than reference IdeSBX1001865. A mean score for fold change in potency relative to referenceIdeS BX1001865 was calculated from all of the scores achieved at thedifferent concentrations for each tested polypeptide, provided that theyfell within the linear section of the calibration curve. This mean scorewas then compared to the mean score achieved for pCART124 referenceIdeS, which was included on each plate to enable comparison betweenplates. The mean score for pCART124 is divided by the mean score for thetest polypeptide to produce a “pCART124 ratio”, which is effectively thefold change in potency relative to IdeS for each polypeptide. ThispCART124 ratio could then be visualised on a bar diagram.

Briefing summarising the laboratory protocol: Wells of multi-titreplates were coated overnight with BSA (10 μg/ml), then washed with PBS-Tand blocked for 1 hour with 2% fish skin gelatine in PBS. IdeS BX1001865polypeptide was prepared as a titration series in 1:2 dilution steps inPBS with 0.1% gelatine from 320 nM down to 0.16 nM. The testpolypeptides and the pCART124 control were then prepared at each of 15,7.5, 3.75, and 1.9 nM in PBS with 0.1% gelatine. A 50 μl sample ofpolypeptide was added to each well with 50 μl of rabbit anti-BSA (ACRIS,# R1048P, 10 nM) as substrate. The plates were incubated at roomtemperature for 1 hour and then washed with PBS-T. Biotinylated goatanti-rabbit Fc-specific antibody (30 000× diluted) was added as adetector antibody and incubated for 30 min. The plate was washed and 40000× diluted SA-Horseradish Peroxidase (HRP; Pierce) was added andincubated for 30 min. The plates were washed and developed using TMB OneComponent as a chromogenic substrate for HRP for 7 min, stopped with 0.5M H₂SO₄. Absorbance (OD) was measured at λ=450 nm. Mean scores for foldchange in potency relative to BX1001865 were determined for each testpolypeptide and for pCART124. The “pCART124 ratio” for each testpolypeptide was then calculated as set out above.

The “pCART124 ratio” results for pCART152, 183, 184, 185, 186, 187, 189and 190 are shown in FIG. 1, alongside the result for pCART124. All ofthe exemplary polypeptides of the invention shown here achieve at least1.5 fold improvement in potency relative to the IdeS control (pCART124).pCART152, 183, 184, 188, 189 and 190 all achieve much higher potency,even as high as 8.0 fold improvement over control for pCART189. Each ofthese six text polypeptides includes the N130R/K modification. ThepCART125 polypeptide was tested separately and achieved potencycomparable to that of IdeS (data not shown).

Visualisation of IgG Cleavage Patterns

The efficacy of the different pCART polypeptides was further evaluatedby visualising on SDS-PAGE the cleavage products produced by a titrationseries of each polypeptide in different substrates. To test efficacy inpure IgG substrate, adalimumab (Humira) was used for IgG1 and denosumab(XGEVA) for IgG2. To test efficacy in a more complex physiologicalenvironment, some of the polypeptides were also titrated in in IVIg(Octagam). This allows the evaluation of the impact of neutralizinganti-IdeS antibodies on polypeptide activity. Cleavage patterns for eachpolypeptide are compared with the cleavage patterns of IdeS (BX1001865and pCART124) in the same substrate. The protocol was follows:

For the pure IgG tests, each test polypeptide or control was diluted ina 1:3 steps titration series from 6.7 μg/ml down to 0.04 ng/ml in PBSwith 0.05% BSA as supporting protein. 25 μl of each concentration wastransferred to multi titre plates and the cleavage reaction was startingby adding 25 μl of either Humira or XGEVA (2 mg/ml). Thus each startingconcentration of polypeptide is diluted 1:2 in the well, giving atitration series of 3.3 μg/ml down to 0.02 ng/ml.

For the IVIg tests, each test polypeptide or control was diluted in a1:2 steps titration series from 30 μg/ml down to 0.015 ng/ml in PBS with0.05% BSA as supporting protein. 25 μl of each concentration wastransferred to multi titre plates and the cleavage reaction was startingby adding 25 μl of 10 mg/ml IVIg. Thus each starting concentration ofpolypeptide is diluted 1:2 in the well, giving a titration series of 15μg/ml down to 0.0075 ng/ml.

The plates were incubated in 37° C. for 1.5 hours. The samples weremixed 1:4 in 2×SDS loading buffer and heated at 92° C. for 5 min 10 μlwere loaded on a polyacrylamide gel (15-well 4-20% Mini-PROTEAN®TGX™precast gel (Biorad) which was read according to standard protocols.

FIG. 2 shows the cleavage patterns produced with IgG1 (adalimumab)substrate for pCART183, 184, 185, 186, 187, 189 and 190 as compared toIdeS (pCART124). Polypeptide concentrations are from 3.33 μg/ml (lane 1)down to 0.02 ng/ml (lane 12) in a 1:3 step dilution series. Intactadalimumab (without enzyme) is visualized to the left (lane C).

The arrows on the right indicate the different cleavage products fromIgG. Arrow 1: Intact IgG; arrow 2: scIgG (single cleaved IgG resultsfrom cleavage of first IgG heavy chain); arrow 3: F(ab′)₂ fragment(results from cleavage of second IgG heavy chain). Vertical lines wereadded to facilitate the comparison at the 1^(st) IgG heavy chaincleavage, where Intact IgG becomes scIgG (between lane 6 and 7) and atthe 2^(nd) IgG heavy chain cleavage, where scIgG becomes F(ab′)₂fragment (between lane 2 and 3).

pCART183, 184, 188, 189 and 190 in particular all show increasedcleavage efficacy of the 2^(nd) heavy chain resulting in more intenseF(ab′)₂ (arrow 3) band and less intense scIgG band (arrow 2), comparedto pCART124 at the same concentrations. The difference is seen from the0.12 μg/ml concentration (lane 4) but is even more evident at 0.37 μg/ml(lane 3). Thus, overall FIG. 2 shows that a change to a positive aminoacid at position 130 (N130R/K) increase the efficacy of cleavage of the2^(nd) IgG heavy chain (pCART183, 184, 188, 189 and 190).

FIG. 3 shows the cleavage patterns produced with IgG1 substrate forpCART152 and pCART209, as compared to both IdeS controls (pCART124 andBX1001865). Enzyme concentrations are from 3.33 μg/ml (lane 1) down to0.02 ng/ml (line 12) in a 1:3 step dilution series. The arrows on theright indicate the different cleavage products from IgG. Arrow 1: IntactIgG; arrow 2: scIgG (single cleaved IgG results from cleavage of firstIgG heavy chain); arrow 3: F(ab′)₂ fragment (results from cleavage ofsecond IgG heavy chain); arrow 4: Fc fragment; (*) indicates the carrierprotein (BSA) in the titration buffer. Vertical lines were added tofacilitate the comparison at the 1^(st) IgG heavy chain cleavage, whereIntact IgG becomes scIgG (between lane 6 and 7) and at the 2^(nd) IgGheavy chain cleavage, where scIgG becomes F(ab′)2 fragment (between lane2 and 3).

pCART152 and pCART209 both show increased cleavage efficacy of the 2ndheavy chain resulting in a more intense F(ab′)₂ bands (arrow 3) and lessscIgG remaining (arrow 2), compared to pCART124 and BX1001865 (arrow 3and 2) at the same concentration. This difference is seen from enzymeconcentrations of 41 ng/ml (lane 5) but is even more evident at 0.12μg/ml and 0.37 μg/ml (lane 4 and 3). Both pCART152 and pCART209 showcleavage efficacy of the 2nd heavy chain about 3× original IdeS(BX1001865 and pCART124), i.e. 0.37 μg/ml of pCART152 and pCART209 (lane3) produces similar result to 1.11 μg/ml of BX100186 and pCART124 (lane2).

pCART152 and pCART209 also show an improved efficacy in the cleavage ofthe 1^(st) IgG heavy chain, resulting in a more intense scIgG band(arrow 2) and a less intense IgG band (arrow 1) compared to originalIdeS (BX1001865 and pCART124) at the same concentration. For pCART152this can be seen at 1.5 ng/ml of enzyme (lane 8) but is even moreobvious at 4.6 ng/ml (lane 7). pCART209 shows an increased cleavageefficacy about 3× original IdeS (BX1001865 and pCART124) also for thefirst heavy chain cleavage, i.e. 4.6 ng/ml (lane 7) of pCART209 producessimilar result to 14 ng/ml (lane 6) of BX1001865 and pCART124.

Thus, FIG. 3 shows that a single amino acid substitution at position 130(N130K in pCART152) increases the efficacy of cleavage of primarily the2^(nd) but also to some extent the 1^(st) IgG1 heavy chain, and that adeletion of the NQTN sequence (pCART209) improves the cleavage efficacyby 3 fold for both the 1^(st) and 2^(nd) IgG1 heavy chains.

FIG. 4 shows the cleavage patterns produced with IgG2 substrate forpCART152 and pCART209, as compared to both IdeS controls (pCART124 andBX1001865). Enzyme concentrations are from 3.33 μg/ml (lane 1) down to0.02 ng/ml (line 12) in a 1:3 step dilution series. The arrows on theright indicate the different cleavage products from IgG. Arrow 1: IntactIgG; arrow 2: scIgG (single cleaved IgG results from cleavage of firstIgG heavy chain); arrow 3: F(ab′)₂ fragment (results from cleavage ofsecond IgG heavy chain); arrow 4: Fc fragment; (*) indicates the carrierprotein (BSA) in the titration buffer. Vertical lines were added tofacilitate the comparison at the 1^(st) IgG heavy chain cleavage, whereIntact IgG becomes scIgG (between lane 6 and 7) and at the 2^(nd) IgGheavy chain cleavage, where scIgG becomes F(ab′)2 fragment (between lane2 and 3).

pCART152 and pCART209 both show increased cleavage efficacy of the2^(nd) heavy chain resulting in a more intense F(ab′)₂ (arrow 3) bandsand less intense scIgG (arrow 2) compared to pCART124 and BX1001865(arrow 3 and 2) at the same concentrations. This difference is seen fromenzyme concentrations of 0.37 μg/ml (lane 3) but even more evident at1.11 μg/ml (lane 2).

The result in lane 6 (enzyme concentration 14 ng/ml) for pCART209 ascompared to BX1001865 and pCART124 shows that pCART209 is also moreeffective at cleaving the 1^(st) IgG heavy chain. This results in a lessintense intact IgG band (arrow 1) and a more intense scIgG band (arrow2) as most protein has been converted to scIgG.

Thus, FIG. 4 shows that a single amino acid substitution at position 130(N130K in pCART152) increases the efficacy of cleavage of primarily the2^(nd) IgG2 heavy chain, and that a deletion of the NQTN sequence(pCART209) improves the cleavage efficacy of both the 1^(st) and 2^(nd)heavy IgG2 chains.

Overall, it can be seen that the test polypeptides are generally moreeffective than IdeS at cleaving IgG1 and IgG2. Consistent with the ELISAresults, pCART152, 183, 184, 188, 189 and 190 appear to be particularlyeffective. The increased efficacy for these polypeptides appears torelate primarily to cleavage of the second IgG heavy chain.

A change to a positive amino acid from the G at position 131 is expectedto produce similar results to the N130R/K substitution. Positions 130and 131 are situated in the loop of a beta hairpin structure spanningpositions 126 to 136 of SEQ ID NO: 1. Based on the results obtainedherein, changes to positive amino acids in either or both of positions130 and 131 are expected to increase IgG cysteine protease activity.

Example 3—Assessment of Immunogenicity Competitive ADA Assay

This assay is based on competition between a test polypeptide and IdeSfor binding to anti-IdeS antibody. A pre-incubation of test enzyme andIVIg will enable binding of anti-IdeS antibodies to the tested pCARTenzyme. Thereafter the IVIg-enzyme-mix is added to an IdeS-coated plateand any anti-IdeS antibody not bound to test polypeptide will insteadbind to the IdeS on the plate. All binding incubations was made in thepresence of 2 mM iodoacetic acid (IHAc) to inhibit IgG cleavage and inhigh salt so that only high affinity binding occurs. After washing, abiotinylated goat anti-human F(ab′)2-specific F(ab′)2 fragment is usedas detector. Poor recognition of test polypeptide by the anti-IdeSantibodies in IVIg will result in high binding of the anti-IdeSantibodies in IVIg to the plate, giving a high signal. Good recognitionof test polypeptide by the anti-IdeS antibodies in IVIg will give theopposition result. The detailed protocol is as follows:

Reference IdeS (BX1001865) was coated overnight on multi-titre plates (5μg/ml), then washed with PBS-T and blocked for 1 hour with 2% BSA in PBSsupplemented with 2 mM IHAc and 1 M NaCl. A mixing plate was preparedwith stepwise dilutions of test polypeptide and 20 μg/ml IVIg in PBSsupplemented with 0.1% BSA, 2 mM IHAc and 1 M NaCl. The mixing plate wasincubated for 1 hour at room temperature on a shaker. After incubation,the blocking solution was discarded from the IdeS-coated plate and 50 μlof each mixture from the mixing plate was transferred to the wells ofthe coated plate. After incubation for 1 hour room temperature on ashaker, the plate was washed with PBS-T and a detector, biotinylatedgoat anti-human F(ab′)₂-specific F(ab′)₂ fragment (20 000× diluted) wasadded. After incubation for 30 minutes the plate was washed and 40 000×diluted SA-HRP (Pierce) was added and incubated for 30 min. The platewas washed and developed using TMB One Component as a chromogenicsubstrate for HRP for 7 min, stopped with 0.5 M H₂SO₄. Absorbance (OD)was measured at λ=450 nm. The results were inverted (1/OD value) andpresented as a ratio compared with pCART124 (1/(testpolypeptide/pCART124)) for visualisation in bar diagrams.

The results for pCART183, 184, 185, 186, 187, 189 and 190 are shown inFIG. 5. The results for pCART125 are shown in FIG. 6. All of the testedpolypeptides show some reduction in anti-IdeS antibody recognition ascompared to IdeS, typically at least 10%. pCART185, 186 and 187 inparticular show a greater reduction in recognition, of around 40%relative to IdeS. pCART125 shows a reduction of around 60%.

SUMMARY

All of the tested polypeptides show increased potency and/or reducedimmunogenicity relative to IdeS.

Example 4—Assessment of Potency Potency ELISA

To address the cleavage capacity of human IgG1 and IgG2, two ELISA-basedpotency assays were set up. One assay measuring IgG1 cleavage and theother IgG2 cleavage. EC50 (half maximal effective concentration) valueswere calculated for the different IgG cysteine protease polypeptidestested. The principle of the assays was to coat wells of a multi titreplate with a F(ab)₂-fragment directed to human IgG antibodies withspecificity to the Fab region. Then titrated concentrations of IgGcysteine protease polypeptide (test or control) were incubated togetherwith human IgG1 antibody (Humira) or human IgG2 antibody (XGEVA) in thewells. The quantity of intact or single cleaved human IgG (Humira orXGEVA) bound to the wells was measured using a detector antibodydirected to human IgG with specificity against the Fc part of theantibody. The higher the concentration of a given IgG cysteine proteasepolypeptide in a well, the less intact human IgG antibody will be boundto the well, giving a lower signal. Similarly, a more potent IgGcysteine protease polypeptide will give a lower signal than a lesspotent IgG cysteine protease polypeptide when present at the sameconcentration. Titration dose-response curves were prepared for the IdeScontrol (pCART124) and all tested IgG cysteine protease polypeptides, inboth the IgG1 (humira) and IgG2 (XGEVA) assay. EC50 values were alsocalculated for each tested variant, representing the concentration of apolypeptide where 50% of its maximal effect, in the second cleavage ofthe IgG molecule, is observed i.e. the concentration where half of theIgG are fully cleaved. A lower EC50 value represents a more effectiveIgG cysteine protease. The cleavage of the first IgG heavy chain, IgG toscIgG, is not visible in this assay because the Fc-part of the IgG isstill present and can be detected by the Fc specific detector antibody(FIG. 13).

Brief summary of the laboratory protocol: Wells of multi titre plateswere coated overnight (+2-8° C.) with Goat-anti-human Fab-specificF(ab)₂-fragment (0.5 μg/ml) (Jackson #109-006-097), then washed withPBS+0.05% Tween 20 (PBS-T) and blocked in 0.45% fish gelatin inPhosphate Buffered Saline-Tween (PBS-T) (block buffer) for 45-120 min atroom temperature. Control IdeS (pCART124) and the IgG cysteine proteasepolypeptides to be tested were prepared as a titration series in 1:4dilution steps in block buffer with a starting concentration of 80μg/ml. Equal volumes (25 μl) of human IgG1 (Humira) at a concentrationof 0.5 μg/ml and the titrated amounts of IgG cysteine proteasepolypeptides were added to the wells and incubated 2 hours with shakingin a controlled temperature environment at 37° C. and then washed withPBS-T. Biotinylated mouse anti-human IgG Fc-specific (m-a-hIgG Bio II,Lot: C0013-ZC43C, Southern Biotech) (600 ng/ml) antibody was mixed withStrep-sulfo (200 ng/ml) and added to the multi titre plates. The plateswere sealed with aluminum tape and incubated at +25° C. for 1 hour withshaking. The plates were then washed in PBS-T and 150 μl of 2× dilutedRead buffer T (MSD read buffer T, Cat. no. R92TC-2) were added to eachwell. The plates were immediately read on a Plate reader, MSD (MesoScale Discovery) QuickPlex SQ 120 Model 1300.

Efficacy assays visualised on gel: Assay conducted as described inExample 2 for cleavage of IgG1 (Humira), IgG2 (XGEVA) as well ascleavage of a pool of human IgG IVIg (Octagam).

Results Potency ELISA

The resulting dose-response curves for the tested IgG cysteine proteasesin the potency assays are shown in FIG. 7 (IgG1 cleavage) and FIG. 8(IgG2 cleavage). pCART125, 213 and 214 of the exemplary polypeptides ofthe invention tested here have improved potency with decreased EC50values (table 1) in cleaving both heavy chains of IgG1 (FIG. 7) and IgG2(FIG. 8) compared to the IdeS control pCART124, with a fold improvementin potency in cutting IgG1 of 1.5 for pCART125, 2.2 for pCART214 and asmuch as 3.0 for pCART213. For cleavage of IgG2 (FIG. 8) the foldimprovement compared to pCART124 (IdeS) was 1.6 for pCART125, 2.1 forpCART214 and 3.5 for pCART213.

Efficacy Assays Visualised on Gel

The cleavage of IgG1 (FIG. 9A) and IgG2 (FIG. 9B) visualised on gelclearly show the first and second heavy chain cleavage (the verticallines in the figures mark the 1^(st) and 2^(nd) IgG heavy chain cleavageby BX1001865 and pCART124 cleavage). The * in the figures illustrate theapproximate EC50 value i.e. the concentration where 50% of the IgG issingle cleaved (scIgG) and 50% is fully cleaved (F(ab′)₂). The data fromthe gels are summarised in table 2 (IgG1 cleavage) and table 3 (IgG2cleavage). The concentration of IgG cysteine protease needed forcleavage of the Pt heavy chain of IgG1 (Humira) is about the same forall polypeptides tested, 1.5 ng/ml for BX1001865 and pCART124 (IdeScontrols), pCART125, 213 and 214 (FIG. 9A). For the 2^(nd) heavy chaincleavage of IgG1 IdeS, pCART125 and 214 approximately 120 ng/ml isneeded do get a dominant F(ab′)2 band on the gels and about onetitration step less, 40 ng/ml, for pCART213 (FIG. 9A). The IgG2 (XGEVA)cleavage visualised in FIG. 9B show that all the polypeptides tested,BX1001865, pCART124, 125, 213 and 214, demonstrate about the sameefficacy with 14 ng/ml needed for cutting the 1^(st) IgG heavy chain andgenerate scIgG2 and about 370 ng/ml to generate F(ab′)₂ fragments (table3). In another set of efficacy experiment pCART228 was tested againstHumira (IgG1) and XGEVA (IgG2) with BX1001865 and pCART124 as IdeScontrols (FIGS. 10A and 10B, table 4). Approximately one titration stephigher concentration is needed for the controls (BX1001865 and pCART124)in these experiments compared to FIG. 9 and table 3, small differencesbetween experiments can be observed due to sample handling andlaboratory precision in the titration dilutions of the samples. To get areliable comparison pCART228 must be compared to the cleavage of IdeScontrols in the same experiment. Very similar IgG cleavage patterns areseen for pCART228 and the IdeS controls in both the IgG1 and IgG2cleavage (FIGS. 10A and B), but with approximately 3× (one titrationstep) more efficient cleavage of the 2^(nd) IgG heavy chain of IgG1 bypCART228 (FIG. 10A) with 120 ng/ml needed compared to 370 ng/ml for IdeS(table 4).

The IgG cysteine protease polypeptides pCART125, 213, and 214 were alsotitrated in the human IgG pool, IVIg (Octagam) with IdeS (BX1001865 andpCART124) as controls (FIG. 11). All tested polypeptides needed 1.5μg/ml for the first IgG heavy chain cleavage and about 6 μg/ml for thesecond (FIG. 11 and table 5). The cleavage of IVIg by pCART228 wasanalysed in a broader titration spectra with 1:2 dilutions from 30 μg/ml(FIG. 12) compared to the tested variants in FIG. 11. The same efficacyis seen for IdeS (BX1001865 and pCART124) and pCART228 (FIG. 12) with aconcentration of 1.9 μg/ml to generate scIgG and 7.5 μg/ml to giveF(ab′)2 fragments (table 6) these represents the same titration steps asfor pCART125, 213 and 214 (FIG. 11 and table 5).

Summary of figures for Example 4

FIG. 7 Titration curves for cleavage of IgG1 (Humira) by different IgGcysteine protease polypeptides.

FIG. 8 Titration curves for cleavage of IgG2 (XGEVA) by different IgGcysteine protease polypeptides.

FIG. 9 IgG cleavage analyzed by SDS-PAGE using titrated (1:3 dilutionfrom 3300 ng/ml) amounts of pCART125, 213 and 214, with BX1001865 andpCART124 (original IdeS) as controls in the same cleavage experiment. A:cleavage of humira (IgG1) and B: cleavage of XGEVA (IgG2). Verticallines mark the IdeS (BX1001865 and pCART124) concentrations needed togive the 1^(st) and 2^(nd) IgG heavy chain cleavage (where the amount ofthe cleaved product dominates over the uncleaved product). The * in thefigures represent the approximate EC50 value in this experiment.

FIG. 10 IgG cleavage analyzed by SDS-PAGE using titrated (1:3 dilutionfrom 3300 ng/ml) amounts of pCART228 with BX1001865 and pCART124(original IdeS) as controls in the same cleavage experiment. A: cleavageof humira (IgG1) and B: cleavage of XGEVA (IgG2). Vertical lines markthe IdeS (BX1001865 and pCART124) concentrations needed to give the1^(st) and 2^(nd) IgG heavy chain cleavage (where the amount of thecleaved product dominates over the uncleaved product).

FIG. 11 IVIg cleavage analyzed by SDS-PAGE using titrated (1:2 dilutionfrom 6 μg/ml) amounts of the tested IgG cysteine protease polypeptidesand IdeS (BX1001865 and pCART124) as control in the same cleavageexperiment.

FIG. 12 IVIg cleavage analyzed by SDS-PAGE using titrated amounts (1:2dilution from 30 μg/ml) of pCART228 with IdeS (BX1001865 and pCART124)as controls in the same cleavage experiment.

FIG. 13 Schematic representation of the cleavage of immunoglobulins bypolypeptides of the invention. The enzymatic cleavage of the IgG isperformed in two steps. First, one heavy chain of intact IgG is cleavedand single cleaved IgG (scIgG) is generated. Secondly, the next IgGheavy chain is cut and the Fc-part is released. The Fc-part is stillattached to the Fab-part in the scIgG molecule and since the detectorantibody in the potency ELISA is recognizing the Fc-part of the IgGmolecule the assay will not differentiate between complete IgG fromscIgG.

Discussion and Conclusion

The lower EC50 values for pCART125, 213 and 214 in the potency ELISAindicate that these polypeptides have an improved potency in the 2^(nd)heavy chain cleavage (from scIgG to F(ab′)₂) of both IgG1 and IgG2compared to pCART124 (original IdeS). Visualising the IgG cleavage ongel shows the cutting of the 1^(st) heavy chain (from IgG to scIgG)which is not measurable in the potency ELISA using an Fc-specificdetector antibody. Most Fc-mediated actions of IgG are lost in a singlecleaved molecule (data not shown), which is central in a clinicalsituation where the main focus is to incapacitate pathogenic IgGmolecules. pCART228 is as effective in both IgG1 and IgG2 cleavage asoriginal IdeS. This is important when an IgG cysteine protease of thesame molecular size as IdeS is needed having the desirable lowerrecognition of IdeS specific antibodies. IVIg is a pool of human IgGcontaining approximately 65-70% IgG1, 35-30% IgG2 and IgG3/IgG4 sharingabout 1%. Human IVIg also naturally contains anti-IdeS antibodies, fromthe IgG donor's earlier exposure to S. pyogenes. The anti-IdeSantibodies in human IVIg are neutralizing insofar as their binding toIdeS may diminish or completely demolish the IdeS IgG protease activity.The results of IVIg cleavage show the overall cleavage of all differentIgG subclasses, with a dominance of IgG1 and IgG2, and with the presenceof neutralizing anti-IdeS antibodies. Generally, all IgG cysteineprotease polypeptides tested have lower efficacy in IgG2 cleavagecompared to IgG1.

TABLE 1 EC50 (ng/ml) measured by potency ELISA and fold difference inpotency compared to original IdeS (pCART124). EC50 EC50 (ng/ml) (ng/ml)in cleavage Fold in cleavage Fold of IgG1 improvement of IgG2improvement (Humira) in potency (XGEVA) in potency pCART124 243 1 199 1pCART125 165 1.5 125 1.6 pCART213 80 3.0 57 3.5 pCART214 111 2.2 95 2.1

TABLE 2 Data for IgG1 (Humira) cleavage shown on gel (FIG. 9A).Concentration (ng/ml) of polypeptide needed to achieve 1^(st) and 2^(nd)IgG cleavage, where the cleaved product dominates in amounts over theuncleaved. Approximate EC50 value (* in FIG. 9A). Approximate EC50value, i.e. equal 1^(st) IgG heavy chain 2^(nd) IgG heavy chain amountsof scIgG and IgG to scIgG scIgG to F(ab′)2 F(ab′)2 (*) Conc. of enzymeConc. of enzyme Conc. of enzyme ID (ng/ml) (ng/ml) (ng/ml) BX1001865 1.5120 100 pCART124 1.5 120 100 pCART125 1.5 120 40-100 pCART213 1.5 40 40pCART214 1.5 120 40-100

TABLE 3 Data for IgG2 (XGEVA) cleavage shown on gel (FIG. 9B).Concentration (ng/ml) of polypeptide needed to achieve 1^(st) and 2^(nd)IgG cleavage, where the cleaved product dominates in amounts over theuncleaved. Approximate EC50 value (* in FIG. 9B). Approximate EC50value, i.e. equal 1^(st) IgG heavy chain 2^(nd) IgG heavy chain amountsof scIgG and IgG to scIgG scIgG to F(ab′)2 F(ab′)2 (*) Conc. of enzymeConc. of enzyme Conc. of enzyme ID (ng/ml) (ng/ml) (ng/ml) BX1001865 14370 100-400 pCART124 14 370 100-400 pCART125 14 370 100-400 pCART213 14370 100-400 pCART214 14 370 100-400

TABLE 4 Data for IgG1 (Humira) cleavage and IgG2 (XGEVA) by pCART228shown on gel (FIG. 10). Concentration (ng/ml) of polypeptide needed toachieve 1^(st) and 2^(nd) IgG cleavage (where the cleaved productdominates in amounts over the uncleaved). 1^(st) IgG1 2^(nd) IgG1 1^(st)IgG2 2^(nd) IgG2 (Humira) heavy (Humira) heavy (XGEVA) heavy (XGEVA)heavy chain IgG to chain scIgG to chain IgG to chain scIgG to scIgGF(ab′)2 scIgG F(ab′)2 Conc. of enzyme Conc. of enzyme Conc. of enzymeConc. of enzyme ID (ng/ml) (ng/ml) (ng/ml) (ng/ml) BX1001865 4.6 370 141100 pCART124 4.6 370 14 1100 pCART228 4.6 120 40 1100

TABLE 5 Data for IVIg cleavage by pCART125, 213 and 214 shown on gel(FIG. 11). Concentration (ng/ml) of polypeptide needed to achieve 1^(st)and 2^(nd) IgG cleavage, where the cleaved product dominates in amountsover the uncleaved. 1^(st) IgG heavy chain 2^(nd) IgG heavy chain IgG toscIgG scIgG to F(ab′)2 Conc. of enzyme Conc. of enzyme ID (ng/ml)(ng/ml) BX1001865 1500 6000 pCART124 1500 6000 pCART125 1500 6000pCART213 1500 6000 pCART214 1500 6000

TABLE 6 Data for IVIg cleavage by pCART228 shown on gel (FIG. 12).Concentration (ng/ml) of polypeptide needed to achieve 1^(st) and 2^(nd)IgG heavy chain cleavage, where the cleaved product dominates in amountsover the uncleaved. 1^(st) IgG heavy chain 2^(nd) IgG heavy chain IgG toscIgG to F(ab')2 Conc. of enzyme Conc. of enzyme ID (ng/ml) (ng/ml)BX1001865 1900 7500 pCART124 1900 7500 pCART229 1900 7500

Example 5—ADA ELISA, a Competitive ELISA for ADA-IdeS Binding Sites

Anti-drug antibody (ADA) binding sites against IdeS was measured for“ADA” modified polypeptide of the invention (pCART125, 213 and 214),using an ELISA, Meso Scale Discovery (MSD), based assay. The principleof the ELISA was to coat wells of a multi titre plate with originalhis-tagged-IdeS (pCART124). Most humans have antibodies against IdeS intheir serum due to earlier infections of group A streptococcus. Here,two different clinical human serum pools were used as standards fordetection of ADA. The first pool is normal human serum from 100individuals, called Human serum pool 1191807, and the second is a poolof serum from patients in the phase II study 13-HMedIdeS-02, calledPhase II pool-2. These patients have been administered with IdeS once ina dose-range of 0.24-0.5 mg/kg body weight and thereby have inducedlevels (approximately 50 times) of anti-IdeS ADA in their serum.

The outline of this competitive ADA ELISA is that IdeS (pCART124) iscoated in the bottom of a micro titre plate. Human serum pools arepre-incubated together with the polypeptides of the invention, to betested for ADA recognition, or with the positive control IdeS (pCART124)in a molar ration of 1:100 with 100× excess of polypeptides of theinvention. The concentration of the two different serum pools used forpre-incubation is estimated from the standard curve to giveapproximately 80% binding to original IdeS. If the ADA binding siteshave been abolished in the polypeptides tested, these variants could notcompete with the binding of ADA to the original IdeS at the bottom ofthe wells, i.e. a low signal demonstrates strong ADA-resemblance to theoriginal IdeS (pCART124) and a high signal demonstrates weakADA-resemblance to the original IdeS.

The concentration of both standards achieving approximately 80% bindingat the linear section of the standard curve was about 200 ng ADA(IdeS)/ml. In the competitive pre-incubation this concentration of bothstandards were used separately and the concentration of the polypeptidesof the invention were used in a concentration of 100 times the ADAconcentration, including the molar weight difference between an antibodyof 150 kDa and IdeS of approximately 35 kDa, 4.2 times, giving 100 times200 ng/ml dividing with 4.2 giving approx. 5 μg/ml of the testedpolypeptides. The standard serum containing 200 ng/ml ADA and the IdeS(pCART124) or polypeptides of the invention are pre-incubated togetherfor 1 hour at room temperature (RT). As a control for maximum ADAbinding, the same concentration of the standards were pre-incubatedwithout IdeS (pCART124) or tested polypeptides and used as 80% bindingmax value. The lowest level of the standards curve, were used as lowerlimit values for the range of the calculation of the competition. Themean score for the standards pre-incubated with IdeS (pCART124) ortested polypeptides were subtracted with the 80% standard binding valuedivided with 80% standard binding value subtracted with the lower limitvalues giving % competition value. The tested polypeptides with thelowest % competition means that the most ADA binding epitopes have beenabolished compared to original IdeS (pCART124).

Brief summary of the laboratory protocol: Wells of multi titre plateswere coated overnight with pCART124 (1 μg/ml), washed 3 times with PBS-Tand blocked for 1 hour with 0.45% fish skin gelatine and 2 mM of thecysteine protease inhibitor Iodoacetic acid (IHAc) in PBS.

Both standards were prepared as titration series in 1:3 dilution stepsin 0.45% fish skin gelatine and 2 mM IHAc in PBS, from 5000 ng ADA(IdeS)/ml to 2.5 ng ADA (IdeS)/ml to allow plotting of a standardcalibration curve for the assay, with measurements at both the linearpart and the maximum and minimum part of the standard curve. At the sametime as the blocking of the plate, the standards and the IdeS (pCART124)or tested polypeptides were pre-incubated together for 1 hour at RT,i.e. the samples in a competition step, using 200 ng/ml ADA (standards)and 5 μg/mlIdeS (pCART124) or polypeptides to be tested.

The pCART124 coated plate was washed 3 times and 50 μl pre-incubatedsamples or 50 μl standard were added to each well of the multi titreplate.

The plate was incubated at RT for 2 hours and then washed with PBS-T.Goat-anti-human F(ab) specific F(ab)₂ fragment-bio (Jackson#109-066-097, 0.65 mg/ml), (1000× diluted) was added as detectorantibody and Streptavidin-Sulfo (MSD Cat. No: R32AD-1 or R32AD-5) (2000×diluted) in blocking buffer incubated for 1 hour at RT in the dark. Theplate was washed 3 times and Read buffer T (MSD Read buffer T (4×) 4×diluted was added and the plate was analysed on a Plate reader, MSD(Meso Scale Discovery) QuickPlex SQ 120 Model 1300 directly.

Results and Conclusion

All tested IgG cysteine protease polypeptides, pCART125, 213 and 214 areless recognized by IdeS specific ADA in human serum compared to originalIdeS (pCART124). Some of the ADA recognition epitopes are found in theN-terminal part of IdeS. All these polypeptides of the invention areN-terminally deleted compared to IdeS and the only sequential differencebetween pCART125 and the original IdeS is the N-terminal deletion.

Percentage (%) blocking of IdeS-ADA binding sites for pCART125, 213 and214 are shown in FIGS. 14 and 15 and the original IdeS pCART124 is usedas positive control for 100% resemblance.

Example 6—Assessment of In Vivo Efficacy in an Octagam (Human IVIg)Mouse Model

In the present study BALB/c mice were injected intraperitoneally (i.p.)with human IVIg (Octagam). The concentration of human IVIg wasadministered at a dose of 900 mg/kg, to correlate to the human IgGplasma concentration (10 mg/ml). Human IVIg was injected i.p. day 0.Twenty four hours (day 1) after the injection of human IVIg, PBS, IdeScontrols (BX1001865 and pCART124) or the IgG cysteine proteases to betested, pCART125, pCART213 and pCART214, were administered intravenously(i.v.) at a dose of 1 mg/kg. Two hours later serum samples werecollected and mice were sacrificed.

Efficacy ELISA

The principle of the assay was to coat wells of a multi titre plate witha F(ab′)2-fragment directed to human IgG antibodies with specificity tothe Fab region. Then serum from mice treated with IVIg and IdeS controls(BX1001865 and pCART124) or the tested IgG cysteine protease polypeptidewere added. The quantity of intact or single cleaved human IgG (IVIg)bound to the wells was measured using a detector antibody directed athuman IgG (IVIg) with specificity against the Fc part of the antibody.The lower the detected concentration of intact human IgG antibody (IVIg)the more effective the IgG cysteine protease polypeptide is expected tobe.

Brief summary of the laboratory protocol: Wells of a multi titre platewere coated overnight (+2-8° C.) with Goat-anti-human Fab-specificF(ab)₂-fragment (0.5 μg/ml) (Jackson #109-006-097), then washed withPBS+0.05% Tween 20 (PBS-T) and blocked in 2% BSA in PBS-T (block buffer)for 45-120 min at RT (room temperature). The Human Serum ProteinCalibrator (DAKO # X0908) was used as a standard and added in a rangefrom 0.5-300 ng/ml. The serum samples taken from mice treated with IVIgand different IgG cysteine protease polypeptides were thawed and dilutedin block buffer 100 000 times before addition to the assay multi titreplate. The plate was incubated 2 hours with shaking at RT and thenwashed with PBS-T. Biotinylated mouse anti-human IgG Fc-specific (600ng/ml) (Jackson #109-066-098) antibody was mixed with Strep-sulfo (200ng/ml) (MSD # R32AD-1) and added to the multi titre plate. The plate wassealed with aluminum tape and incubated at RT for 1 hour with shaking.The plate was then washed in PBS-T and 150 μl of 2× diluted Read bufferT (MSD # R92TC-2) was added to each well. The plate was immediatelyanalysed on a plate reader, MSD (Meso Scale Discovery) QuickPlex SQ 120Model 1300 directly.

Efficacy Visualized on Gel

10 μl mice serum was diluted in 1:10 in 90 μl PBS. Thereafter 10 μldiluted serum was mixed with 30 μl 4×SDS-PAGE loading buffer. 5 μl ofIgG in-house marker was used to show the different IgG fragments (IgG,scIgG and F(ab′)2). Samples were heated at 92° C. for 3 min (Thermomixer compact, eppendorf) and briefly centrifuged before loading 10 μlon 4-20% Mini-Protean® TGX, Stain-Free™ gel (Cat. #456-8096, Biorad).Gels were run at 200 V for 40 min

Results and Conclusion

In vivo cleavage of human IVIg (Octagam) by IdeS controls (BX1001865 andpCART124) and pCART125, 213 and 214 were compared by studying the levelof human IgG in serum by efficacy ELISA and by analysing the degradationof IgG by SDS-PAGE. Treatment with the IdeS controls (BX1001865 andpCART124) and the different IgG cysteine proteases pCART125, pCART213and pCART214 in mice showed a clear effect on cleaving human IgG in vivo(Table 7 and FIG. 16).

Complete cleavage were shown for the IdeS controls (BX100186 andpCART124), pCART125 and pCART213, with no scIgG bands visible andsignificant F(ab′)₂ bands on the gels (FIG. 17). pCART214 showed a lowerefficacy in this mouse model with scIgG molecules present in the serumafter two hours (* in FIG. 17C). However, no intact IVIg could bedetected on the gel meaning that the higher bar for pCART214 in FIG. 16represents scIgG and not intact IgG. This shows that polypeptides of theinvention cleave IgG in an in vivo model.

TABLE 7 Analysis of in vivo cleavage of human IgG in serum from micetreated with IdeS (BX1001865 and pCART124)/IdeS variants by the efficacyELISA (average ± Stdev). Average (mg/mL) Stdev Control (PBS) 6.58 0.80BX1001865 0.30 0.05 pCART124 0.39 0.12 pCART125 0.38 0.39 pCART213 0.260.06 pCART214 1.29 0.05

1. A polypeptide having IgG cysteine protease activity and comprising a variant of the sequence of SEQ ID NO:2, which variant: (a) is at least 50% identical to SEQ ID NO: 2; (b) has a cysteine (C) at the position in said variant sequence which corresponds to position 94 of SEQ ID NO: 1; and optionally (c) has, at the positions in said variant sequence which correspond to positions 84, 262, 284 and 286 of SEQ ID NO: 1, a lysine (K), a histidine (H), an aspartic acid (D) and an aspartic acid (D), respectively; wherein said polypeptide is more effective at cleaving IgG than IdeS and/or is less immunogenic than IdeS.
 2. A polypeptide according to claim 1, wherein said variant of the sequence of SEQ ID NO: 2: (1) has a positively charged amino acid at the position in said variant which corresponds to position 130 of SEQ ID NO: 1, optionally wherein said positively charged amino acid is arginine (R) or lysine (K); and/or (2) has a positively charged amino acid at the position in said variant which corresponds to position 131 of SEQ ID NO: 1, optionally wherein said positively charged amino acid is arginine (R) or lysine (K); and/or (3) does not include the contiguous sequence NQTN; and/or (4) does not include the contiguous sequence DSFSANQEIR YSEVTPYHVT.
 3. A polypeptide according to claim 1 or 2, wherein said variant of the sequence of SEQ ID NO: 2 is at least 80%, 90%, 95% or 99% identical to SEQ ID NO:
 2. 4. A polypeptide according to any one of the preceding claims, which comprises or consists of the sequence of any one of SEQ ID NOs: 3 to 16, optionally wherein said sequence includes an additional methionine at the N terminus and/or a histidine tag at the C terminus.
 5. A polypeptide according to any one of the preceding claims, wherein said polypeptide is at least 1.5 fold, 2.0 fold, 2.5 fold, 3.0 fold, 4.0 fold, 4.5 fold, 5.0 fold, 6.0 fold, 7.0 fold or 7.5 fold greater more effective than IdeS at cleaving IgG, when measured in the same assay.
 6. A polypeptide according to any one of the preceding claims which is less immunogenic than IdeS, wherein preferably the immunogenicity of said polypeptide is no more than 85% of the immunogenicity of IdeS when measured in the same assay.
 7. A polynucleotide or expression vector which comprises a nucleic acid sequence encoding a polypeptide of any one of the preceding claims.
 8. A host cell comprising the polynucleotide or expression vector of claim 7, which is preferably bacterial cell, most preferable a cell of E. coli.
 9. A composition comprising a polypeptide according to any one of claims 1 to 6 and at least one pharmaceutically acceptable carrier or diluent.
 10. A polypeptide according to any one of claims 1 to 6 for use in the treatment of the human or animal body.
 11. A method for the prevention or treatment of a disease or condition in a subject, which method comprises administering to the subject a polypeptide according to any one of claims 1 to 6 to the subject in a prophylactically or therapeutically effective amount.
 12. A method according to claim 11, wherein said disease or condition is a disease or condition mediated in whole or in part by pathogenic IgG antibodies, preferably wherein said disease or condition is listed in Table D.
 13. A method for the cleavage of IgG, the method comprising contacting a sample containing IgG with a polypeptide according to any one of claims 1 to 6 under conditions which permit IgG cysteine protease activity to occur.
 14. A method according to claim 13 which is carried out ex vivo and/or is conducted to generate Fc and Fab fragments and/or wherein the sample is a blood sample taken from a subject suffering from a disease or condition as defined in claim
 12. 